SIRPalpha-41BBL FUSION PROTEIN AND METHODS OF USE THEREOF

ABSTRACT

SIRP1alpha-41BBL fusion proteins are provided. Accordingly, there is provided a SIRPalpha-41BBL fusion protein comprising a single amino acid linker between the SIRPalpha and the 41BBL. Also there is provided a SIRPalpha-41BBL fusion protein in a form of at least a homo-trimer. Also provided are polynucleotides and nucleic acid constructs encoding the SIRP1alpha-41BBL fusion protein, host-cells expressing the SIRP1alpha-41BBL fusion protein and methods of use thereof.

BACKGROUND OF THE INVENTION

Dual Signaling Proteins (DSP), also known as Signal-Converting-Proteins(SCP), which are currently known in the art as bi-functional fusionproteins that link an extracellular portion of a type I membrane protein(extracellular amino-terminus), to an extracellular portion of a type IImembrane protein (extracellular carboxyl-terminus), forming a fusionprotein with two active sides (see, for example, U.S. Pat. Nos.7,569,663 and 8,039,437, both of which are hereby incorporated byreference as if fully set forth herein).

SIRPα (signal-regulatory protein alpha) is a cell surface receptor ofthe immunoglobulin superfamily. SIRPα is expressed mainly on the surfaceof immune cells from the phagocyte lineage like macrophages anddendritic cells (DC). CD47 is the ligand of SIRPα. CD47 is a cellsurface molecule in the immunoglobulin superfamily. CD47 functions as aninhibitor of phagocytosis through ligation of SIRPα expressed onphagocytes. CD47 is widely expressed on a majority of normal tissues. Inthis way, CD47 serves as a “don't eat me signal” and a marker of self,as loss of CD47 leads to homeostatic phagocytosis of aged or damagedcells. CD47 has been found to be expressed on multiple human tumortypes. Tumors evade macrophage phagocytosis through the expression ofantiphagocytic signals, including CD47. While CD47 is ubiquitouslyexpressed at low levels on normal cells, multiple tumors expressincreased levels of CD47 compared to their normal cell counterparts andover-expression of CD47 enabled tumors to escape innate immune systemsurveillance through evasion of phagocytosis. 4-1BBL is the activatingligand of the 41BB receptor (CD137), a member of the TNF receptorsuperfamily and a potent activation-induced T cell costimulatorymolecule. 41BBL naturally forms a homo-trimer but signaling via 4-1BBrequires significant oligomerization of 4-1BBL. 4-1BBL is present on avariety of antigen presenting cells (APCs), including dendritic cells(DCs), B cells, and macrophages. The 4-1BB receptor is not detected(<3%) on resting T cells or T cell lines, however, 4-1BB is stablyupregulated when T cells are activated. 4-1BB activation upregulatessurvival genes, enhances cell division, induces cytokine production andprevents activation induced cell death in T-cells.

Additional background art includes:

International Patent Application Publication No. WO2017059168;

International Patent Application Publication No. WO2001/049318;

International Patent Application Publication No. WO2016/139668;

International Patent Application Publication No. WO2014/106839;

International Patent Application Publication No. WO2012/042480;

US Patent Application Publication No. 20150183881;

US Patent Application Publication No. US20070110746;

US Patent Application Publication No. US20070036783; and

U.S. Pat. No. 9,562,087.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a SIRPα-41BBL fusion protein comprising a single aminoacid linker between the SIRPα and the 41BBL.

According to an aspect of some embodiments of the present inventionthere is provided a SIRPα-41BBL fusion protein in a form of at least ahomo-trimer.

According to some embodiments of the invention, the at least homo-trimeris at least 140 kD in molecular weight as determined by SDS-PAGE.

According to some embodiments of the invention, the SIRPα-41BBL fusionprotein comprises a linker between the SIRPα and the 41BBL.

According to some embodiments of the invention, the linker has a lengthof one to six amino acids.

According to some embodiments of the invention, the linker is a singleamino acid linker.

According to some embodiments of the invention, the linker is not an Fcdomain of an antibody or a fragment thereof.

According to some embodiments of the invention, the linker is glycine.

According to some embodiments of the invention, the SIRPα-41BBL fusionprotein being soluble.

According to some embodiments of the invention, the SIRPα comprises anextracellular domain of the SIRPα or a functional fragment thereof.

According to some embodiments of the invention, the 41BBL comprises anextracellular domain of the 41BBL or a functional fragment thereof.

According to some embodiments of the invention, the fusion protein iscapable of at least one of:

-   -   (i) binding CD47 and 41BB;    -   (ii) activating the 41BB signaling pathway in a cell expressing        the 41BB;    -   (iii) co-stimulating immune cells expressing the 41BB; and/or    -   (iv) enhancing phagocytosis of pathologic cells expressing the        CD47 by phagocytes compared to same in the absence of the        SIRPα-41BBL fusion protein.

According to some embodiments of the invention, the SIRPα-41BBL fusionprotein amino acid sequence comprises SEQ ID NO: 1.

According to some embodiments of the invention, the SIRPα-41BBL fusionprotein amino acid sequence consists of SEQ ID NO: 1.

According to some embodiments of the invention, there is provided apolynucleotide encoding the SIRPα-41BBL fusion protein of the presentinvention.

According to some embodiments of the invention, there is provided anucleic acid construct comprising the polynucleotide of the presentinvention, and a regulatory element for directing expression of thepolynucleotide in a host cell.

According to some embodiments of the invention, the polynucleotidecomprises SEQ ID NO: 8.

According to some embodiments of the invention, there is provided a hostcell comprising the SIRPα-41BBL fusion protein of the present inventionor the polynucleotide or the nucleic acid construct of the presentinvention.

According to some embodiments of the invention, there is provided amethod of producing a SIRPα-41BBL fusion protein, the method comprisingexpressing in a host cell the polynucleotide or the nucleic acidconstruct of the present invention.

According to some embodiments of the invention, the method comprisingisolating the fusion protein.

According to some embodiments of the invention, the cell is selectedfrom the group consisting of CHO, PERC.6 and 293.

According to some embodiments of the invention, there is provided amethod of treating cancer comprising administering the SIRPα-41BBLfusion protein of the present invention to a subject in need thereof.

According to some embodiments of the invention, there is provided amethod of treating a disease that can benefit from activating immunecells comprising administering to a subject in need thereof theSIRPα-41BBL fusion protein of the present invention, the polynucleotideor the nucleic acid construct of the present invention or the host cellof any one of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture identified for the treatmentof a disease that can benefit from activating immune cells comprising apackaging material packaging a therapeutic agent for treating thedisease; and a SIRPα-41BBL fusion protein, a polynucleotide encodingsame, a nucleic acid construct encoding same or a host cell expressingsame.

According to some embodiments of the invention, the disease comprises ahyper-proliferative disease.

According to some embodiments of the invention, the hyper-proliferativedisease comprises sclerosis or fibrosis, Idiopathic pulmonary fibrosis,psoriasis, systemic sclerosis/scleroderma, primary biliary cholangitis,primary sclerosing cholangitis, liver fibrosis, prevention ofradiation-induced pulmonary fibrosis, myelofibrosis or retroperitonealfibrosis.

According to some embodiments of the invention, the hyper-proliferativedisease comprises cancer.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer comprising administeringto a subject in need thereof an anti-cancer agent; and a SIRPα-41BBLfusion protein, a polynucleotide encoding same, a nucleic acid constructencoding same or a host cell expressing same.

According to some embodiments of the invention, the cancer is selectedfrom the group consisting of lymphoma, leukemia, colon cancer,pancreatic cancer, ovarian cancer, lung cancer and squamous cellcarcinoma.

According to some embodiments of the invention, the cells of the cancerexpress CD47.

According to some embodiments of the invention, the disease comprises adisease associated with immune suppression or medication inducedimmunosuppression.

According to some embodiments of the invention, the disease comprisesHIV, Measles, influenza, LCCM, RSV, Human Rhinoviruses, EBV, CMV orParvo viruses.

According to some embodiments of the invention, the disease comprises aninfection.

According to some embodiments of the invention, diseased cells of thesubject express CD47.

According to an aspect of some embodiments of the present inventionthere is provided a method of activating T cells, the method comprisingin-vitro activating T cells in the presence of a SIRPα-41BBL fusionprotein and cells expressing CD47.

According to an aspect of some embodiments of the present inventionthere is provided a method of activating phagocytes, the methodcomprising in-vitro activating phagocytes in the presence of aSIRPα-41BBL fusion protein and cells expressing CD47.

According to an aspect of some embodiments of the present inventionthere is provided a method of activating immune cells, the methodcomprising in-vitro activating immune cells in the presence of aSIRPα-41BBL fusion protein, a polynucleotide encoding same, a nucleicacid construct encoding same or a host cell expressing same.

According to some embodiments of the invention, the activating is in thepresence of cells expressing CD47 or exogenous CD47.

According to some embodiments of the invention, the cells expressing theCD47 comprise pathologic cells.

According to some embodiments of the invention, the pathologic cellscomprise cancer cells.

According to some embodiments of the invention, the cancer is selectedfrom the group consisting of lymphoma, carcinoma and leukemia.

According to some embodiments of the invention, the activating is in thepresence of an anti-cancer agent.

According to some embodiments of the invention, the anti-cancer agentcomprises an antibody.

According to some embodiments of the invention, the antibody is selectedfrom the group consisting rituximab, cetuximab, trastuzumab,edrecolomab, almetuzumab, gemtuzumab, ibritumomab, panitumumab,Belimumab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab,Blontuvetmab, Brentuximab vedotin, Catumaxomab, Cixutumumab, Daclizumab,Adalimumab, Bezlotoxumab, Certolizumab pegol, Citatuzumab bogatox,Daratumumab, Dinutuximab, Elotuzumab, Ertumaxomab, Etaracizumab,Gemtuzumab ozogamicin, Girentuximab, Necitumumab, Obinutuzumab,Ofatumumab, Pertuzumab, Ramucirumab, Siltuximab, Tositumomab,Trastuzumab and ipilimumab.

According to some embodiments of the invention, the antibody is selectedfrom the group consisting of rituximab, cetuximab and alemtuzumab.

According to some embodiments of the invention, the method comprisingadoptively transferring the immune cells following the activating to asubject in need thereof.

According to some embodiments of the invention, the subject is afflictedwith a disease associated with the cells expressing the CD47.

According to some embodiments of the invention, the SIRPα-41BBL fusionprotein comprises the SIRPα-41BBL fusion protein of the presentinvention, the polynucleotide or the nucleic acid construct comprisesthe polynucleotide or the nucleic acid construct of the presentinvention, and the host cell comprises the host cell of the presentinvention.

According to some embodiments of the invention, the immune cellscomprise T cells.

According to some embodiments of the invention, the immune cellscomprise phagocytes.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a photograph of western blot analysis of His-taggedSIRPα-41BBL (SEQ ID NO: 5) under reducing or non-reducing conditions.Following affinity purification, proteins (250 ng/well) were separatedon SDS-PAGE gel under denaturing or non denaturing conditions, asindicated, followed by immunoblotting with an anti-His-tag antibody.

FIGS. 2A-B are photographs of western blot analysis of His-taggedSIRPα-41BBL (SEQ ID NO: 5) under reducing or non-reducing conditions.Following affinity purification, proteins (250 ng/well) were separatedon SDS-PAGE gel under denaturing (FIG. 2A) or non-denaturing (FIG. 2B)conditions, followed by immunoblotting with an anti-41BBL antibody.

FIG. 2C is a photograph of coomassie blue staining of SDS-PAGE analysisof His-tagged SIRPα-41BBL (SEQ ID NO: 5) under reducing conditionstreated or un-treated with de-glycosylase. His-tagged SIRPα-41BBL bandsare marked with small black arrows.

FIGS. 3A-B are graphs demonstrating interaction of His-taggedSIRPα-41BBL (SEQ ID NO: 5) with its counterpart ligands, as determinedby bio-layer interferometry Blitz® assay.

FIG. 3A demonstrates binding to CD47—the biosensor was pre-loaded withCD47:Fc and then incubated with His-tagged SIRPα-41BBL (SEQ ID NO: 5) orPD1-CD70 (SEQ ID NO: 6, as a negative control). FIG. 3B demonstratesbinding to 41BB—the biosensor was pre-loaded with 41BB:Fc and thenincubated with His-tagged SIRPα-41BBL (SEQ ID NO: 5) or PD1-CD70 (SEQ IDNO: 6, as a negative control).

FIGS. 4A-B are histograms demonstrating expression patterns of theindicated receptors on CHO-K1-WT cells (FIG. 4A) and CHO-KI-CD47 cells(FIG. 4B). The surface expression levels of 41BB and CD47 was determinedby immuno-staining of each cell line with the corresponding antibodies,followed by flow cytometric analysis.

FIGS. 5A-B demonstrate binding of His-tagged SIRPα-41BBL protein (SEQ IDNO: 5) to CHO-K1-47 cells (FIG. 5A) but not to CHO-K1-WT cells (FIG.5B). The cells were incubated with different concentrations ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) for 30 minutes, followedby immune-staining with anti-41BBL antibody and flow cytometry analysis.GMFI values were used to create a binding curve graph with a GraphPadPrism software.

FIG. 6 is a graph demonstrating that His-tagged SIRPα-41BBL protein (SEQID NO: 5) promotes TNFR signaling as demonstrated by IL-8 secretion fromHT1080-41BB cells in medium containing FBS.

FIG. 7 is a graph demonstrating that His-tagged SIRPα-41BBL protein (SEQID NO: 5) promotes TNFR signaling as demonstrated by IL-8 secretion fromHT1080-41BB cells in serum free media.

FIGS. 8A-E demonstrate that His-tagged SIRPα-41BBL protein (SEQ ID NO:5) triggers 41BB co-stimulatory signaling and potentiates T cellactivation. FIG. 8A is a graph demonstrating IL-8 concentration insupernatant of HT1080-41BB cell cultures and co-cultures of HT1080-41BBand CHO cells following treatment with His-tagged SIRPα-41BBL protein(SEQ ID NO: 5). FIG. 8B is a graph demonstrating IL-8 concentration insupernatant of HT1080-41BB cell cultures and co-cultures of HT1080-41BBand CHO-CD47 cells following treatment with His-tagged SIRPα-41BBLprotein (SEQ ID NO: 5). FIG. 8C is a graph demonstrating T cellsactivation, as evaluated by CD25 expression, in T cells cultured for 3days in 96-wells plates coated with CD47:Fc and treated with asub-optimal concentration of anti-CD3/anti-CD38 beads and increasingconcentrations of His-tagged SIRPα-41BBL (SEQ ID NO: 5). FIGS. 8D-Eshows representative dot plots (FIG. 8D) and a summarizing graph(mean±standard error, two independent donors were taken in twoindependent experiments. Each donor was analyzed in triplicates. FIG. 8Eare dot plots demonstrating T cells activation, as evaluated by CD25expression, in co-cultures of T cells isolated from peripheral blood ofhealthy volunteers mixed with DLD-1 cells and treated for 3 days with asub-optimal concentration of anti-CD3/anti-CD28 beads with or withoutHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5).

FIGS. 9A-C demonstrate that SIRPα-41BBL does not have a direct killingeffect on MV4-11 cancer cells. FIG. 9A is a histogram demonstrating CD47expression on the surface of MV4-11 cells, FIG. 9B demonstrates bindingof His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) to MV4-11 cells. Thecells were incubated with Fc-blocker for 15 minutes on ice followed byincubation with different concentrations of His-tagged SIRPα-41BBLprotein (SEQ ID NO: 5) for 30 minutes, immuno-staining with anti-41BBLantibody and flow cytometry analysis. FIG. 9C is a graph showing thatincubation of MV4-11 cells with different concentrations of His-taggedSIRPα-41BBL protein (SEQ ID NO: 5) for up to 72 hours did not show anydirect killing effect, as determined by PI staining.

FIGS. 10A-B demonstrate that SIRPα-41BBL promotes INF-γ secretion fromanti-CD3 primed human PBMCs. FIG. 10A is a graph demonstrating IFN-γconcentration detected in the culture supernatant of human PBMCsincubated for 40 hours with different concentrations of His-taggedSIRPα-41BBL protein (SEQ ID NO: 5) in the presence of anti-CD3 oranti-CD3 plus IL2, as indicated. FIG. 10B is a graph demonstrating IFN-γconcentration detected in the culture supernatant of human PBMCsco-cultured with human cancer MV-4-11 cells and incubated for 40 hourswith different concentrations of His-tagged SIRPα-41BBL protein (SEQ IDNO: 5), with or without anti-CD3; and with or without IL2, as indicated.

FIGS. 11A-L demonstrate that SIRPα-41BBL potentiatesgranulocyte-mediated phagocytosis. FIG. 11A shows a representativegating strategy of the flow cytometric phagocytosis analysis. FIG. 11Bis a graph demonstrating phagocytosis of B-cell lymphoma cell line BJABby granulocytes following treatment with the indicated concentrations ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11C is a graphdemonstrating phagocytosis of the indicated B-cell lymphoma cell linesby granulocytes obtained from 2-3 individual donors incubated with orwithout His-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11D is agraph demonstrating phagocytosis of the indicated B-cell lymphoma celllines by granulocytes following treatment with rituximab with or withoutHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11E is a graphdemonstrating phagocytosis of the indicated carcinoma cell lines bygranulocytes obtained from 3-6 individual donors incubated with orwithout His-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11F is agraph demonstrating phagocytosis of the indicated carcinoma cell linesby granulocytes following treatment with cetuximab with or withoutHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11G is a graphdemonstrating phagocytosis of the indicated myeloid leukemia cell linesby granulocytes obtained from 3 individual donors incubated for 2 hourswith or without His-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11His a graph demonstrating phagocytosis of the indicated myeloid leukemiacell lines by granulocytes obtained from 3 individual donors incubatedfor 24 hours with or without His-tagged SIRPα-41BBL protein (SEQ ID NO:5). FIG. 11I is a graph demonstrating phagocytosis of HL60 and MOLM13 bygranulocytes following treatment with the indicated concentrations ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 11J is a graphdemonstrating phagocytosis of primary acute myeloid leukaemia cells bygranulocytes following 2 hours or 24 hours treatment with the indicatedconcentrations of His-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG.11K is a graph demonstrating phagocytosis (mean±standard error) ofprimary acute myeloid leukaemia cells by allogeneic granulocytesobtained from 5 individual donors following 2 hours or 24 hoursincubation with or without 2.5 μg/ml His-tagged SIRPα-41BBL protein (SEQID NO: 5). FIG. 11L shows phagocytosis of DLD1 cancer cells bypolymorphonuclear cells treated for 2 hours with soluble SIRPα, soluble41BBL, combination of both or His tagged-SIRPα-41BBL fusion protein (SEQID NO: 5).

FIGS. 12A-D demonstrate that SIRPα-41BBL potentiates macrophage-mediatedphagocytosis. FIG. 12A shows representative microscopic pictures ofco-cultures of macrophages and B-cell lymphoma cell line U2932pre-stained V450 following 2 hours of incubation with or withoutHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) and with or without themonoclonal antibody rituximab (mAb). Adhered macrophages are visible inbright-field, with V450-labelled cancer cells being visible as brightcells. Dark arrows indicate viable tumour cells. White arrows indicatetumor cells that have been phagocytosed by macrophages.

FIG. 12B is a graph demonstrating macrophage-mediated phagocytosis ofU2932 cells following treatment with the indicated concentrations ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5). FIG. 12C is a graphdemonstrating macrophage-mediated phagocytosis of U2932 cells followingtreatment with rituximab with or without His-tagged SIRPα-41BBL protein(SEQ ID NO: 5). FIG. 12D is a graph demonstrating macrophage-inducedphagocytosis (mean±standard error) of primary B-cell malignant chroniclymphocytic leukaemia incubated with or without His-tagged SIRPα-41BBLprotein (SEQ ID NO: 5) and with or without alemtuzumab, FIGS. 13A-Bdemonstrate that treatment of CT-26 inoculated mice with His-taggedSIRPα-41BBL protein (SEQ ID NO: 5) reduces tumor volume FIG. 13A is aschematic illustration of experiment timelines: mice were inoculatedS.C. with 1×10⁶ CT-26 cells on day 0, PBS control, aPD1 or SIRPα-41BBLwere injected on days 3, 7 and 10. FIG. 13B is a graph demonstratingmean±standard error) tumor volume in the three treatment groups.

FIGS. 14A-B demonstrate that His-tagged SIRPα-41BBL protein (SEQ ID NO:5) is effective for the treatment of mice inoculated with P388 syngeneicleukemia tumor. FIG. 14A is a schematic illustration of experimenttimelines: mice were inoculated I.P. with 1×10⁶ P388 cells on day 0, PBScontrol, aPD1 or SIRPα-41BBL were injected on days 1, 3, 5, and 7. FIG.14B is a graph demonstrating spleen weight in the three treatment groupsupon sacrifice.

FIGS. 15A-C demonstrate that His-tagged SIRPα-41BBL protein (SEQ ID NO:5) decreases tumor burden in the BM of NSG mice inoculated with humanleukemia tumor. FIG. 15A is a schematic illustration of experimenttimelines: mice were irradiated 24 hr before inoculation of MV4.11cells, thirteen (13) days later mice were inoculated with human PBMCs,treatment started 4 hours later. 5 animals per group were administeredevery-other-day (EOD) injections with four intraperitoneal of His-taggedSIRPα-41BBL protein (100 μg/injection) or its soluble buffer (PBS) (ondays 13, 15, 17 and 19). Twenty-four (24) hours after the last injectionmice were sacrificed. FIG. 15B shows the number of leukemic cells in theBM as was determined using flow cytometry. FIG. 15C shows spleen weightin mg.

DESCRIPTION OF DETAILED EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to aSIRPα-41BBL fusion protein and methods of use thereof.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Dual Signaling Proteins (DSP), also known as Signal-Converting-Proteins(SCP), which are currently known in the art as bi-functional fusionproteins that link an extracellular portion of a type I membrane protein(extracellular amino-terminus), to an extracellular portion of a type IImembrane protein (extracellular carboxyl-terminus), forming a fusionprotein with two active sides.

Surprisingly, it was found that a specific fusion protein may beadvantageously administered to subjects suffering from cancerousdiseases, depending upon the presence of tumors that havetumor-infiltrating lymphocytes (TILs) in the tumor micro-environment aswell as tumors with relatively high expression of CD47 on the tumorcells or in the tumor micro-environment.

As is illustrated hereinunder and in the examples section, whichfollows, the present inventors have produced a his-tagged SIRPα-41BBLfusion protein (SEQ ID NO: 5) and show that the fusion protein (SEQ IDNO: 5) contains both domains and produced in the form of at leasttrimers (Experiments 1A-B, FIGS. 1 and 2A-C). Following, the presentinventors demonstrate that the produced his-tagged SIRPα-41BBL fusionprotein (SEQ ID NO: 5) retains functional binding activity for itscognate receptors CD47 and 41BB (Experiments 1C-D, FIGS. 3A-B, 4A-B and5A-B) and can trigger 41BB co-stimulation and activation of cellsexpressing 41BB (e.g. T cells, PBMCs) wherein presence of CD47 augmentsthis activity (Experiments 2-3 and 3A-B, FIGS. 6-7, 8A-E 9A-C and10A-B). In addition, the inventors demonstrate that the His-taggedSIRPα-41BBL (SEQ ID NO: 5) through its SIRPα domain augments phagocyticuptake of various malignant cell types, including primary malignantcells, particularly in combination treatment with various therapeuticmonoclonal antibodies currently in clinical use (Experiment 4 FIGS.11A-L and 12A-D). The inventors further demonstrate that his-taggedSIRPα-41BBL fusion protein (SEQ ID NO: 5) is effective for the treatmentof tumors as shown in in-vivo colon carcinoma and leukemia mouse tumormodels (Experiments 5 and 5A-C, FIGS. 13A-B, 14A-B and 15A-C).

Consequently, the present teachings suggest SIRPα-41BBL fusion proteins,polynucleotides encoding same and host cells expressing same; and usesof same in e.g. activating immune cells (via co-stimulation) in generaland treating diseases that can benefit from activating immune cells(e.g. cancer) in particular.

Thus, according to a first aspect of the present invention, there isprovided a SIRPα-41BBL fusion protein or any variants or fragmentsthereof or a SIRPα-41BBL fusion protein, which is at least about 70%,homologous to the sequence as set forth in SEQ ID No. 4 optionally witha linker therebetween.

According to another aspect of the present invention, there is provideda SIRPα-41BBL fusion protein comprising a single amino acid linkerbetween said SIRPα and said 41BBL.

According to another aspect of the present invention, there is provideda SIRPα-41BBL fusion protein in a form of at least a homo-trimer.

According to specific embodiments, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95% of the SIRPα-41BBL fusion protein is ina form of at least a homo-trimer, each possibility represents a separateembodiment of the present invention.

According to specific embodiments, the at least homo-trimer comprises ahomo-trimer.

According to specific embodiments, the at least homo-trimer comprises ahomo-tetramer.

According to specific embodiments, the at least homo-trimer comprises ahomo-pentamer.

According to specific embodiments, the at least homo-trimer comprises ahomo-hexamer.

Methods of determining trimerization are well known in the art andinclude, but are not limited to SDS-PAGE, NATIVE-PAGE, SEC-HPLC, 2Dgels, gel filtration, SEC MALLS, Analytical ultracentrifugation (AUC)Mass spectrometry (MS), capillary gel electrophoresis (CGE).

According to specific embodiments the at least homo-trimer is at least140 kD, at least 160 kD, at least 180 kD at least 200 kD, at least 220kD, at least 240 kD in molecular weight as determined by SDS-PAGE.

According to specific embodiments the at least homo-trimer is at least140 kD in molecular weight as determined by SDS-PAGE.

According to specific embodiments, the at least homo-trimer is at least200 kD in molecular weight as determined by SDS-PAGE.

As used herein the term “SIRPα (Signal Regulatory Protein Alpha, alsoknown as CD172a)” refers to the polypeptide of the SIRP1 gene (Gene ID140885) or a functional homolog e.g., functional fragment thereof.According to specific embodiments, the term “SIRPα” refers to afunctional homolog of SIRPα polypeptide. According to specificembodiments, SIRPα is human SIRPα. According to a specific embodiment,the SIRPα protein refers to the human protein, such as provided in thefollowing GenBank Number NP_005009.

As used herein, a “functional SIRPα” is capable of binding its cognatereceptor CD47 [also known as integrin associated protein (IAP)].

As use herein, the phrase “functional homolog” or “functional fragment”when related to SIRPα, refers to a portion of the polypeptide whichmaintains the activity of the full length SIRPα e.g., CD47 binding.

According to a specific embodiment, the CD47 protein refers to the humanprotein, such as provided in the following GenBank Numbers NP_001768 orNP_942088.

Assays for testing binding are well known in the art and include, butnot limited to flow cytometry, BiaCore, bio-layer interferometry Blitz®assay, HPLC.

According to specific embodiments, the SIRPα binds CD47 with a Kd of0.1-100 μM, 0.1-10 μM, 1-10 μM, 0.1-5 μM, or 1-2 μM as determined bySPR, each possibility represented a separate embodiment of the presentinvention.

According to specific embodiments, the SIRPα comprises an extracellulardomain of said SIRPα or a functional fragment thereof.

According to specific embodiments, SIRPα amino acid sequence comprisesSEQ ID NO: 9.

According to specific embodiments, SIRPα amino acid sequence consists ofSEQ ID NO: 9.

According to specific embodiments, SIRPα nucleic acid sequence comprisesSEQ ID NO: 10.

According to specific embodiments, SIRPα nucleic acid sequence consistsof SEQ ID NO: 10.

According to specific embodiments, SIRPα amino acid sequence comprisesSEQ ID NO: 2.

According to specific embodiments, SIRPα amino acid sequence consists ofSEQ ID NO: 2.

According to specific embodiments, SIRPα nucleic acid sequence comprisesSEQ ID NO: 11.

According to specific embodiments, SIRPα nucleic acid sequence consistsof SEQ ID NO: 11.

The term “SIRPα” also encompasses functional homologues (naturallyoccurring or synthetically/recombinantly produced), which exhibit thedesired activity (i.e., binding CD47). Such homologues can be, forexample, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical or homologous tothe polypeptide SEQ ID NO: 2 or 9; or at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to the polynucleotide sequence encoding same (as furtherdescribed hereinbelow).

Sequence identity or homology can be determined using any protein ornucleic acid sequence alignment algorithm such as Blast, ClustalW, andMUSCLE.

The homolog may also refer to an ortholog, a deletion, insertion, orsubstitution variant, including an amino acid substitution, as furtherdescribed hereinbelow.

According to specific embodiments, the SIRPα polypeptide may compriseconservative amino acid substitutions as further described hereinbelow.

According to specific embodiments, SIRPα amino acid sequence comprises100-500 amino acids, 150-450 amino acids, 200-400 amino acids, 250-400amino acids, 300-400 amino acids, 320-420 amino acids, 340-350 aminoacids, each possibility represents a separate embodiment of the presentinvention.

According to specific embodiments, SIRPα amino acid sequence is 300-400amino acids in length.

According to specific embodiments, SIRPα amino acid sequence is 340-450amino acids in length.

According to specific embodiments, SIRPα amino acid sequence is 343amino acids in length.

As used herein the term “41BBL (also known as CD137L and TNFSF9)” refersto the polypeptide of the TNFSF9 gene (Gene ID 8744) or a functionalhomolog e.g., functional fragment thereof. According to specificembodiments, the term “41BBL” refers to a functional homolog of 41BBLpolypeptide. According to specific embodiments, 41BBL is human 41BBL.According to a specific embodiment, the 41BBL protein refers to thehuman protein, such as provided in the following GenBank NumberNP_003802.

According to specific embodiments, the 41BBL comprises an extracellulardomain of said 41BBL or a functional fragment thereof.

According to specific embodiments, 41BBL amino acid sequence comprisesSEQ ID NO: 12.

According to specific embodiments, 41BBL amino acid sequence consists ofSEQ ID NO: 12.

According to specific embodiments, 41BBL nucleic acid sequence comprisesSEQ ID NO: 13.

According to specific embodiments, 41BBL nucleic acid sequence consistsof SEQ ID NO: 13.

According to specific embodiments, 41BBL amino acid sequence comprisesSEQ ID NO: 3.

According to specific embodiments, 41BBL amino acid sequence consists ofSEQ ID NO: 3.

According to specific embodiments, 41BBL nucleic acid sequence comprisesSEQ ID NO: 14.

According to specific embodiments, 41BBL nucleic acid sequence consistsof SEQ ID NO: 14.

The term “41BBL” also encompasses functional homologues (naturallyoccurring or synthetically/recombinantly produced), which exhibit thedesired activity (as defined hereinbelow). Such homologues can be, forexample, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical or homologous tothe polypeptide SEQ ID NO: 3, 13; or at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to the polynucleotide sequence encoding same (as furtherdescribed hereinbelow).

According to specific embodiments, the 41BBL polypeptide may compriseconservative amino acid substitutions, as further described hereinbelow.

According to specific embodiments, 41BBL amino acid sequence comprises100-300 amino acids, 150-250 amino acids, 100-250 amino acids, 150-220amino acids, 180-220 amino acids, 190-210 amino acids, each possibilityrepresents a separate embodiment of the present invention.

According to specific embodiments, 41BBL amino acid sequence is 190-210amino acids in length.

According to specific embodiments, 41BBL amino acid sequence is 204amino acids in length.

As used herein, a “functional 41BBL” is capable of least one of:

-   -   (i) binding its cognate receptor 41BB (also known as CD137),    -   (ii) activating 41BB signaling pathway in an immune cell        expressing 41BB; and/or    -   (iii) activating immune cells expressing said 41BB.

According to specific embodiments, functional 41BBL is capable of (i),(ii), (iii), (i)+(ii), (i)+(iii), (ii)+(iii).

According to specific embodiments, functional 41BBL is capable of(i)+(ii)+(iii).

As use herein, the phrase “functional homolog” or “functional fragment”when related to 41BBL, refers to a portion of the polypeptide whichmaintains the activity of the full length 41BBL e.g., binding 41BB,activating 41BB signaling pathway, activating immune cells expressing41BB.

According to a specific embodiment, the 41BB protein refers to the humanprotein, such as provided in the following GenBank Number NP_001552.

Assays for testing binding are well known in the art and are furtherdescribed hereinabove

According to specific embodiments, the 41BBL binds 41BB with a Kd ofabout 0.1-1000 nM, 0.1-100 nM, 1-100 nM, or 55.2 nM as determined bySPR, each possibility represents a separate embodiment of the claimedinvention.

As used herein the terms “activating” or “activation” refer to theprocess of stimulating an immune cell (e.g. T cell, B cell, NK cell,phagocytic cell) that results in cellular proliferation, maturation,cytokine production, phagocytosis and/or induction of regulatory oreffector functions.

According to specific embodiments, activating comprises co-stimulating.

As used herein the term “co-stimulating” or “co-stimulation” refers totransmitting a secondary antigen independent stimulatory signal (e.g.41BB signal) resulting in activation of the immune cell.

According to specific embodiments, activating comprises suppressing aninhibitory signal (e.g. CD47 signal) resulting in activation of theimmune cell.

Methods of determining signaling of a stimulatory or inhibitory signalare well known in the art and also disclosed in the Examples sectionwhich follows, and include, but are not limited to, binding assay usinge.g. BiaCore, HPLC or flow cytometry, enzymatic activity assays such askinase activity assays, and expression of molecules involved in thesignaling cascade using e.g.

PCR, Western blot, immunoprecipitation and immunohistochemistry.Additionally or alternatively, determining transmission of a signal(co-stimulatory or inhibitory) can be effected by evaluating immune cellactivation or function. Methods of evaluating immune cell activation orfunction are well known in the art and include, but are not limited to,proliferation assays such as CFSE staining, MTS, Alamar blue, BRDU andthymidine incorporation, cytotoxicity assays such as CFSE staining,chromium release, Calcin AM, cytokine secretion assays such asintracellular cytokine staining, ELISPOT and ELISA, expression ofactivation markers such as CD25, CD69, CD137, CD107a, PD1, and CD62Lusing flow cytometry.

According to specific embodiments, determining the signaling activity oractivation is effected in-vitro or ex-vivo e.g. in a mixed lymphocytereaction (MLR), as further described hereinbelow.

For the same culture conditions the signaling activity or the immunecell activation or function are generally expressed in comparison to thesignaling, activation or function in a cell of the same species but notcontacted with the SIRPα-41BBL fusion protein, a polynucleotide encodingsame or a host cell encoding same; or contacted with a vehicle control,also referred to as control.

The terms “DSP” and “fusion protein”, “chimeric protein” or “chimera”are used herein interchangeably, and refer to an amino acid sequencehaving two or more parts which are not found together in a single aminoacid sequence in nature.

In one embodiment, the present invention is directed to a fusion proteincomprising a SIRPα-41BBL, (hereinafter, SIRPα-41BBL fusion protein) orany variants or fragments thereof optionally with a linker therebetween.

SIRPα-41BBL is a Dual Signaling Protein (DSP) chimera protein fusing theextracellular domains of two different human membrane proteins. The Nterminal domain is the extracellular domain of the human SIRPα (gene:SIRPA), which is a type 1 membrane protein, and the C terminal domain ofthe chimera is the extracellular domain of the human 41BBL (gene:41BBL), which is a type 2 membrane protein.

According to specific embodiments, the SIRPα-41BBL fusion protein issoluble (i.e., not immobilized to a synthetic or a naturally occurringsurface).

According to specific embodiments, the SIRPα-41BBL fusion protein isimmobilized to a synthetic or a naturally occurring surface.

According to specific embodiments, the SIRPα-41BBL does not comprise alinker between the SIRPα and the 41BBL.

In some embodiment, the SIRPα-41BBL comprises a linker which may be atany length.

Hence, according to specific embodiments the SIRPα-41BBL fusion proteincomprises a linker between said SIRPα and said 41BBL.

Any linker known in the art can be used with specific embodiments of theinvention.

According to specific embodiments, the linker may be derived fromnaturally-occurring multi-domain proteins or is an empirical linker asdescribed, for example, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, theentire contents of which are hereby incorporated by reference. In someembodiments, the linker may be designed using linker designing databasesand computer programs such as those described in Chen et al., (2013),Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et al (2000), ProteinEng. 13(5):309-312, the entire contents of which are hereby incorporatedby reference.

According to specific embodiments, the linker is a synthetic linker suchas PEG.

According to specific embodiments, the linker is an Fc domain or thehinge region of an antibody (e.g., of IgG, IgA, IgD or IgE) or afragment thereof.

According to other specific embodiments, the linker is not an Fc domainor a hinge region of an antibody or a fragment thereof.

According to specific embodiments, the linker may be functional. Forexample, without limitation, the linker may function to improve thefolding and/or stability, improve the expression, improve thepharmacokinetics, and/or improve the bioactivity of the SIRPα-41BBLfusion protein. In another example, the linker may function to targetthe SIRPα-41BBL fusion protein to a particular cell type or location.

According to specific embodiments, the linker is a polypeptide.

In some embodiments, the SIRPα-41BBL comprises a linker at a length ofone to six amino acids.

According to specific embodiments, the linker is substantially comprisedof glycine and/or serine residues (e.g. about 30%, or about 40%, orabout 50%, or about 60%, or about 70%, or about 80%, or about 90%, orabout 95%, or about 97% or 100% glycines and serines).

According to specific embodiments, the linker is a single amino acidlinker.

In some embodiments of the invention, the amino acid which links SIRPαand 41BBL is glycine, also referred to herein as SIRPα-G-41BBL fusionprotein.

According to specific embodiments, the SIRPα-41BBL fusion protein aminoacid sequence comprises SEQ ID NO: 1.

According to specific embodiments, the SIRPα-41BBL fusion protein aminoacid sequence consists of SEQ ID NO: 1.

In some embodiments, the term “SIRPα-G-41BBL fusion protein” refers to aprotein identified by SEQ ID NO. 1:

Amino-acid sequence of the chimera protein (SIRPα- G-41BBL):EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYGACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

The extracellular domain of the human SIRPα protein is underlined i.e.

(SEQ ID NO. 2) EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSNISTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY

The extracellular domain of the human 41BBL is bold i.e.

(SEQ ID NO. 3) ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSE

According to specific embodiments, the amino acid sequence ofSIRPα-G-41BBL is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% homologous to the amino acid sequence as set forth in SEQ IDNo. 1 or to the polynucleotide sequence encoding same.

In some embodiments, there is provided a SIRPα-41BBL fusion protein,which is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% homologous to the sequence as set forth in SEQ ID No. 4 optionallywith a linker between SIRPα peptide or the ECD thereof and 41BBL peptideor the ECD thereof, wherein SEQ ID No. 4 is:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some embodiments, there is provided a SIRPα-41BBL as set forth in SEQID No. 4 optionally with a linker between SIRPα peptide or the ECDthereof and 41BBL peptide or the ECD thereof, wherein SEQ ID No. 4 is:

EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSNISTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In additional embodiments, the SIRPA-G-41BBL fusion protein may be avariant and/or derivative of the amino acid sequence shown in SEQ IDNO. 1. A number of such variants are known in the art, see as forexample in Weiskopf et al, 2013; Young Won, et al, 2010 and Rabu, et al,2005; Hereby incorporated by reference as if fully set forth herein.

According to specific embodiments, the SIRPα-41BBL fusion protein iscapable of least one of:

-   -   (i) binding CD47 and 41BB,    -   (ii) activating 41BB signaling pathway in an immune cell (e.g. T        cell) expressing 41BB;    -   (iii) activating immune cells (e.g. T cells) expressing said        41BB; and/or    -   (iv) enhancing phagocytosis of pathologic cells expressing CD47        by phagocytes compared to same in the absence of SIRPα-41BBL        fusion protein.

According to specific embodiments, the SIRPα-41BBL fusion protein iscapable of (i), (ii), (iii), (iv), (i)+(ii), (i)+(iii), (i)+(iv),(ii)+(iii), (ii)+(iv), (i)+(ii)+(iii), (i)+(ii)+(iv), (ii)+(iii)+(iv).

According to specific embodiments, the SIRPα-41BBL fusion protein iscapable of (i)+(ii)+(iii)+(iv).

Methods of determining binding, activating 41BB signaling pathway andactivating immune cells are well known in the art and are furtherdescribed hereinabove and below and in the Examples section whichfollows.

According to specific embodiments, the SIRPα-41BBL fusion proteinenhances phagocytosis of pathologic cells expressing CD47 by phagocytes.

Methods of analyzing phagocytosis are well known in the art and are alsodisclosed in Experiment 4 in the Examples section which follows; andinclude for examples killing assays, flow cytometry and/or microscopicevaluation (live cell imaging, fluorescent microscopy confocalmicroscopy, electron microscopy).

According to specific embodiments the enhancement in phagocytosis is atleast 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, atleast 10 fold, or at least 20 fold as compared to same in the absence ofthe SIRPα-41BBL fusion protein, the polynucleotide or nucleic acidconstruct encoding same or the host cell expressing same of the presentinvention, as determined by e.g. flow cytometry or microscopicevaluation.

According to other specific embodiments the increase in survival is byat least 5%, by at least a 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95% or at least 100% as compared to same in the absence ofthe SIRPα-41BBL fusion protein, the polynucleotide or nucleic acidconstruct encoding same or the host cell expressing same of the presentinvention, as determined by e.g. flow cytometry or microscopicevaluation.

As the compositions of some embodiments of present invention (e.g. thefusion protein, a polynucleotide or nucleic acid encoding same or a hostcell expressing same) are capable of activating immune cells, they canbe used in method of activating immune cells, in-vitro, ex-vivo and/orin-vivo.

Thus, according to an aspect of the present invention, there is provideda method of activating immune cells, the method comprising in-vitro orex-vivo activating immune cells in the presence of a SIRPα-41BBL fusionprotein, a polynucleotide encoding same, a nucleic acid constructencoding same or a host cell expressing same.

According to another aspect of the present invention, there is provideda method of activating T cells, the method comprising in-vitro orex-vivo activating T cells in the presence of a SIRPα-41BBL fusionprotein and cells expressing CD47.

According to another aspect of the present invention, there is provideda method of activating phagocytes, the method comprising in-vitroactivating phagocytes in the presence of a SIRPα-41BBL fusion proteinand cells expressing CD47.

According to specific embodiments, the immune cells express 41BB.

According to specific embodiments, the immune cells comprise peripheralmononuclear blood cells (PBMCs).

As used herein the term “peripheral mononuclear blood cells (PBMCs)”refers to a blood cell having a single nucleus and includes lymphocytes,monocytes and dendritic cells (DCs).

According to specific embodiments, the PBMCs are selected from the groupconsisting of dendritic cells (DCs), T cells, B cells, NK cells and NKTcells.

According to specific embodiments, the PBMCs comprise T cells, B cells,NK cells and NKT cells.

Methods of obtaining PBMCs are well known in the art, such as drawingwhole blood from a subject and collection in a container containing ananti-coagulant (e.g. heparin or citrate); and apheresis. Following,according to specific embodiments, at least one type of PBMCs ispurified from the peripheral blood. There are several methods andreagents known to those skilled in the art for purifying PBMCs fromwhole blood such as leukapheresis, sedimentation, density gradientcentrifugation (e.g. ficoll), centrifugal elutriation, fractionation,chemical lysis of e.g. red blood cells (e.g. by ACK), selection ofspecific cell types using cell surface markers (using e.g. FACS sorteror magnetic cell separation techniques such as are commerciallyavailable e.g. from Invitrogen, Stemcell Technologies, Cellpro, AdvancedMagnetics, or Miltenyi Biotec.), and depletion of specific cell types bymethods such as eradication (e.g. killing) with specific antibodies orby affinity based purification based on negative selection (using e.g.magnetic cell separation techniques, FACS sorter and/or capture ELISAlabeling).

Such methods are described for example in THE HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Volumes 1 to 4, (D.N. Weir, editor) and FLOW CYTOMETRY ANDCELL SORTING (A. Radbruch, editor, Springer Verlag, 2000).

According to specific embodiments, the immune cells comprise tumorinfiltrating lymphocytes.

As used herein the term “tumor infiltrating lymphocytes (TILs) refers tomononuclear white blood cells that have lest the bloodstream andmigrated into a tumor.

According to specific embodiments, the TILs are selected from the groupconsisting of T cells, B cells, NK cells and monocytes.

Methods of obtaining TILs are well known in the art, such as obtainingtumor samples from a subject by e.g. biopsy or necropsy and preparing asingle cell suspension thereof. The single cell suspension can beobtained in any suitable manner, e.g., mechanically (disaggregating thetumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn,Calif.) or enzymatically (e.g., collagenase or DNase). Following, the atleast one type of TILs can be purified from the cell suspension. Thereare several methods and reagents known to those skilled in the art forpurifying the desired type of TILs, such as selection of specific celltypes using cell surface markers (using e.g. FACS sorter or magneticcell separation techniques such as are commercially available e.g. fromInvitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, orMiltenyi Biotec.), and depletion of specific cell types by methods suchas eradication (e.g. killing) with specific antibodies or by affinitybased purification based on negative selection (using e.g. magnetic cellseparation techniques, FACS sorter and/or capture ELISA labeling). Suchmethods are described for example in THE HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Volumes 1 to 4, (D.N. Weir, editor) and FLOW CYTOMETRY ANDCELL SORTING (A. Radbruch, editor, Springer Verlag, 2000).

According to specific embodiments, the immune cells comprise phagocytes.

As used herein, the term “phagocytes” refer to a cell that is capable ofphagocytosis and include both professional and non-professionalphagocytes. Methods of analyzing phagocytosis are well known in the artand are further disclosed hereinabove and below. According to specificembodiments, the phagocytic cells are selected from the group consistingof monocytes, dendritic cells (DCs) and granulocytes.

According to specific embodiments, the phagocytes comprise granulocytes.

According to specific embodiments, the phagocytes comprise monocytes.

According to specific embodiments, the immune cells comprise monocytes.

According to specific embodiments, the term “monocytes” refers to bothcirculating monocytes and to macrophages (also referred to asmononuclear phagocytes) present in a tissue.

According to specific embodiments, the monocytes comprise macrophages.Typically, cell surface phenotype of macrophages include CD14, CD40,CD11b, CD64, F4/80 (mice)/EMR1 (human), lysozyme M, MAC-1/MAC-3 andCD68.

According to specific embodiments, the monocytes comprise circulatingmonocytes. Typically, cell surface phenotypes of circulating monocytesinclude CD14 and CD16 (e.g. CD14++CD16-, CD14+CD16++, CD14++CD16+).

According to specific embodiments, the immune cells comprise DCs

As used herein the term “dendritic cells (DCs)” refers to any member ofa diverse population of morphologically similar cell types found inlymphoid or non-lymphoid tissues. DCs are a class of professionalantigen presenting cells, and have a high capacity for sensitizingHLA-restricted T cells. DCs include, for example, plasmacytoid dendriticcells, myeloid dendritic cells (including immature and mature dendriticcells), Langerhans cells, interdigitating cells, follicular dendriticcells. Dendritic cells may be recognized by function, or by phenotype,particularly by cell surface phenotype. These cells are characterized bytheir distinctive morphology having veil-like projections on the cellsurface, intermediate to high levels of surface HLA-class II expressionand ability to present antigen to T cells, particularly to naive T cells(See Steinman R, et al., Ann. Rev. Immunol. 1991; 9:271-196.).Typically, cell surface phenotype of DCs include CDla+, CD4+, CD86+, orHLA-DR. The term DCs encompasses both immature and mature DCs.

According to specific embodiments, the immune cells comprisegranulocytes.

As used herein, the tern “granulocytes” refer to polymorphonuclearleukocytes characterized by the presence of granules in their cytoplasm.

According to specific embodiments, the granulocytes compriseneutrophils.

According to specific embodiments, the granulocytes comprise mast-cells.

According to specific embodiments the immune cells comprise T cells.

As used herein, the term “T cells” refers to a differentiated lymphocytewith a CD3+, T cell receptor (TCR)+ having either CD4+ or CD8+phenotype. The T cell may be either an effector or a regulatory T cell.

As used herein, the term “effector T cells” refers to a T cell thatactivates or directs other immune cells e.g. by producing cytokines orhas a cytotoxic activity e.g., CD4+, Th1/Th2, CD8+ cytotoxic Tlymphocyte.

As used herein, the term “regulatory T cell” or “Treg” refers to a Tcell that negatively regulates the activation of other T cells,including effector T cells, as well as innate immune system cells. Tregcells are characterized by sustained suppression of effector T cellresponses. According to a specific embodiment, the Treg is aCD4+CD25+Foxp3+ T cell.

According to specific embodiments, the T cells are CD4+ T cells.

According to other specific embodiments, the T cells are CD8+ T cells.

According to specific embodiments, the T cells are memory T cells.Non-limiting examples of memory T cells include effector memory CD4+ Tcells with a CD3+/CD4+/CD45RA−/CCR7− phenotype, central memory CD4+ Tcells with a CD3+/CD4+/CD45RA−/CCR7+ phenotype, effector memory CD8+ Tcells with a CD3+/CD8+CD45RA−/CCR7-phenotype and central memory CD8+ Tcells with a CD3+/CD8+CD45RA−/CCR7+ phenotype.

According to specific embodiments, the T cells comprise engineered Tcells transduced with a nucleic acid sequence encoding an expressionproduct of interest.

According to specific embodiments, the expression product of interest isa T cell receptor (TCR) or a chimeric antigen receptor (CAR).

As used herein the phrase “transduced with a nucleic acid sequenceencoding a TCR” or “transducing with a nucleic acid sequence encoding aTCR” refers to cloning of variable co- and β-chains from T cells withspecificity against a desired antigen presented in the context of MHC.Methods of transducing with a TCR are known in the art and are disclosede.g. in Nicholson et al. Adv Hematol. 2012; 2012:404081; Wang andRivibre Cancer Gene Ther. 2015 March; 22(2):85-94); and Lamers et al,Cancer Gene Therapy (2002) 9, 613-623.

As used herein, the phrase “transduced with a nucleic acid sequenceencoding a CAR” or “transducing with a nucleic acid sequence encoding aCAR” refers to cloning of a nucleic acid sequence encoding a chimericantigen receptor (CAR), wherein the CAR comprises an antigen recognitionmoiety and a T-cell activation moiety. A chimeric antigen receptor (CAR)is an artificially constructed hybrid protein or polypeptide containingan antigen binding domain of an antibody (e.g., a single chain variablefragment (scFv)) linked to T-cell signaling or T-cell activationdomains. Method of transducing with a CAR are known in the art and aredisclosed e.g. in Davila et al. Oncoimmunology. 2012 Dec. 1;1(9):1577-1583; Wang and Rivibre Cancer Gene Ther. 2015 March;22(2):85-94); Maus et al. Blood. 2014 Apr. 24; 123(17):2625-35; Porter DL The New England journal of medicine. 2011, 365(8):725-733; Jackson HJ, Nat Rev Clin Oncol. 2016; 13(6):370-383; and Globerson-Levin et al.Mol Ther. 2014; 22(5):1029-1038.

According to specific embodiments, the immune cells comprise B cells.

As used herein the term “B cells” refers to a lymphocyte with a B cellreceptor (BCR)+, CD19+ and or B220+ phenotype. B cells are characterizedby their ability to bind a specific antigen and elicit a humoralresponse.

According to specific embodiments, the immune cells comprise NK cells.

As used herein the term “NK cells” refers to differentiated lymphocyteswith a CD16+CD56+ and/or CD57+ TCR− phenotype. NK are characterized bytheir ability to bind to and kill cells that fail to express “self”MHC/HLA antigens by the activation of specific cytolytic enzymes, theability to kill tumor cells or other diseased cells that express aligand for NK activating receptors, and the ability to release proteinmolecules called cytokines that stimulate or inhibit the immuneresponse.

According to specific embodiments, the immune cells comprise NKT cells.

As used herein the term “NKT cells” refers to a specialized populationof T cells that express a semi-invariant αβ T-cell receptor, but alsoexpress a variety of molecular markers that are typically associatedwith NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1−, aswell as CD4+, CD4−, CD8+ and CD8− cells. The TCR on NKT cells is uniquein that it recognizes glycolipid antigens presented by the MHC I-likemolecule CD1d. NKT cells can have either protective or deleteriouseffects due to their abilities to produce cytokines that promote eitherinflammation or immune tolerance.

According to specific embodiments, the immune cells are obtained from ahealthy subject.

According to specific embodiments, the immune cells are obtained from asubject suffering from a pathology (e.g. cancer).

According to specific embodiments, the activating is in the presence ofcells expressing CD47 or exogenous CD47.

According to specific embodiments, the activating is in the presence ofexogenous CD47, According to specific embodiments, the exogenous CD47 issoluble.

According to other specific embodiments, the exogenous CD47 isimmobilized to a solid support.

According to specific embodiments, the activating is in the presence ofcells expressing CD47.

According to specific embodiments, the cells expressing the CD47comprise pathologic (diseased) cells.

According to specific embodiments, the cells expressing the CD47comprise cancer cells.

According to specific embodiments, the activating is in the presence ofa stimulatory agent capable of at least transmitting a primaryactivating signal [e.g. ligation of the T-Cell Receptor (TCR) with theMajor Histocompatibility Complex (MHC)/peptide complex on the AntigenPresenting Cell (APC)] resulting in cellular proliferation, maturation,cytokine production, phagocytosis and/or induction of regulatory oreffector functions of the immune cell. According to specificembodiments, the stimulator agent can also transmit a secondaryco-stimulatory signal.

Methods of determining the amount of the stimulatory agent and the ratiobetween the stimulatory agent and the immune cells are well within thecapabilities of the skilled in the art and thus are not specifiedherein.

The stimulatory agent can activate the immune cells in anantigen-dependent or -independent (i.e. polyclonal) manner.

According to specific embodiments, stimulatory agent comprises anantigen non-specific stimulator.

Non-specific stimulators are known to the skilled in the art. Thus, as anon-limiting example, when the immune cells comprise T cells, antigennon-specific stimulator can be an agent capable of binding to a T cellsurface structure and induce the polyclonal stimulation of the T cell,such as but not limited to anti-CD3 antibody in combination with aco-stimulatory protein such as anti-CD28 antibody. Other non-limitingexamples include anti-CD2, anti-CD137, anti-CD134, Notch-ligands, e.g.Delta-like 1/4, Jagged1/2 either alone or in various combinations withanti-CD3. Other agents that can induce polyclonal stimulation of T cellsinclude, but not limited to mitogens, PHA, PMA-ionomycin, CEB andCytoStim (Miltenyi Biotech). According to specific embodiments, theantigen non-specific stimulator comprises anti-CD3 and anti-CD28antibodies. According to specific embodiments, the T cell stimulatorcomprises anti-CD3 and anti-CD28 coated beads, such as the CD3CD28MACSiBeads obtained from Miltenyi Biotec.

According to specific embodiments, the stimulatory agent comprises anantigen-specific stimulator.

Non-limiting examples of antigen specific T cell stimulators include anantigen-loaded antigen presenting cell [APC, e.g. dendritic cell] andpeptide loaded recombinant MHC. Thus, for example, a T cells stimulatorcan be a dendritic cell preloaded with a desired antigen (e.g. a tumorantigen) or transfected with mRNA coding for the desired antigen.

According to specific embodiments, the antigen is a cancer antigen.

As used herein, the term “cancer antigen” refers to an antigen isoverexpressed or solely expressed by a cancerous cell as compared to anon-cancerous cell. A cancer antigen may be a known cancer antigen or anew specific antigen that develops in a cancer cell (i.e. neoantigens).

Non-limiting examples for known cancer antigens include MAGE-AI,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9,MAGE-AIO, MAGE-All, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1/CT7, MAGE-C2,NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1and XAGE, melanocyte differentiation antigens, p53, ras, CEA, MUCI,PMSA, PSA, tyrosinase, Melan-A, MART-I, gplOO, gp75, alphaactinin-4,Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a,coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein,LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-All, hsp70-2,KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9, pml-RARalpha fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomerase,GnTV, Herv-K-mel, NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, plSOerbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, alpha.-fetoprotein, 13HCG, BCA225, BTAA,CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1,C0-029, FGF-5, 0250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag,MOV18, NB\170K, NYCO-I, RCASI, SDCCAG16, TA-90 (Mac-2 bindingprotein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS,tyrosinase related proteins, TRP-1, or TRP-2.

Other tumor antigens that may be expressed are well-known in the art(see for example WO00/20581; Cancer Vaccines and Immunotherapy (2000)Eds Stern, Beverley and Carroll, Cambridge University Press, Cambridge).The sequences of these tumor antigens are readily available from publicdatabases but are also found in WO 1992/020356 AI, WO 1994/005304 AI, WO1994/023031 AI, WO 1995/020974 AI, WO 1995/023874 AI & WO 1996/026214AI. Alternatively, or additionally, a tumor antigen may be identifiedusing cancer cells obtained from the subject by e.g. biopsy.

Thus, according to specific embodiments, the stimulatory agent comprisesa cancer cell.

According to specific embodiments, the activating is in the presence ofan anti-cancer agent.

According to specific embodiments, the immune cells are purifiedfollowing the activation.

Thus, the present invention also contemplated isolated immune cellsobtainable according to the methods of the present invention.

According to specific embodiments, the immune cells used and/or obtainedaccording to the present invention can be freshly isolated, stored e.g.,cryopreserved (i.e. frozen) at e.g. liquid nitrogen temperature at anystage for long periods of time (e.g., months, years) for future use; andcell lines.

Methods of cryopreservation are commonly known by one of ordinary skillin the art and are disclosed e.g. in International Patent ApplicationPublication Nos. WO2007054160 and WO 2001039594 and US PatentApplication Publication No. US20120149108.

According to specific embodiments, the cells obtained according to thepresent invention can be stored in a cell bank or a depository orstorage facility.

Consequently, the present teachings further suggest the use of theisolated immune cells and the methods of the present invention as, butnot limited to, a source for adoptive immune cells therapies fordiseases that can benefit from activating immune cells e.g. ahyper-proliferative disease; a disease associated with immunesuppression and infections.

Thus, according to specific embodiments, method of the present inventioncomprise adoptively transferring the immune cells following saidactivating to a subject in need thereof.

According to specific embodiments, there is provided the immune cellsobtainable according to the methods of the present invention are for usein adoptive cell therapy.

The cells used according to specific embodiments of the presentinvention may be autologous or non-autologous; they can be syngeneic ornon-syngeneic: allogeneic or xenogeneic to the subject; each possibilityrepresents a separate embodiment of the present invention.

The present teachings also contemplates the use of the compositions ofthe present invention (e.g. the fusion protein, a polynucleotide ornucleic acid construct encoding same or a host cell expressing same) inmethods of treating a disease that can benefit from activating immunecells.

Thus, according to another aspect of the present invention, there isprovided a method of treating a disease that can benefit from activatingimmune cells comprising administering to a subject in need thereof theSIRPα-41BBL fusion protein, a polynucleotide or nucleic acid constructencoding same or a host cell encoding same.

According to another aspect of the present invention, there is providedthe SIRPα-41BBL fusion protein, a polynucleotide or nucleic acidconstruct encoding same or a host cell encoding same for use in thetreatment of a disease that can benefit from activating immune cells.

The term “treating” or “treatment” refers to inhibiting, preventing orarresting the development of a pathology (disease, disorder or medicalcondition) and/or causing the reduction, remission, or regression of apathology or a symptom of a pathology. Those of skill in the art willunderstand that various methodologies and assays can be used to assessthe development of a pathology, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of apathology.

As used herein, the term “subject” includes mammals, e.g., human beingsat any age and of any gender. According to specific embodiments, theterm “subject” refers to a subject who suffers from the pathology(disease, disorder or medical condition). According to specificembodiments, this term encompasses individuals who are at risk todevelop the pathology.

According to specific embodiments, the subject is afflicted with adisease associated with cells expressing CD47.

According to specific embodiments, diseases cells of the subject expressCD47.

As used herein the phrase “a disease that can benefit from activatingimmune cells” refers to diseases in which the subject's immune responseactivity may be sufficient to at least ameliorate symptoms of thedisease or delay onset of symptoms, however for any reason the activityof the subject's immune response in doing so is less than optimal.

Non-limiting examples of diseases that can benefit from activatingimmune cells include hyper-proliferative diseases, diseases associatedwith immune suppression, immunosuppression caused by medication (e.g.mTOR inhibitors, calcineurin inhibitor, steroids) and infections.

According to specific embodiments, the disease comprises ahyper-proliferative disease.

According to specific embodiments, the hyper-proliferative diseasecomprises sclerosis or fibrosis, Idiopathic pulmonary fibrosis,psoriasis, systemic sclerosis/scleroderma, primary biliary cholangitis,primary sclerosing cholangitis, liver fibrosis, prevention ofradiation-induced pulmonary fibrosis, myelofibrosis or retroperitonealfibrosis.

According to other specific embodiments, the hyper-proliferative diseasecomprises cancer.

Thus, according to another aspect of the present invention, there isprovided a method of treating cancer comprising administering theSIRPα-41BBL fusion protein to a subject in need thereof.

As used herein, the term cancer encompasses both malignant andpre-malignant cancers.

With regard to pre-malignant or benign forms of cancer, optionally thecompositions and methods thereof may be applied for halting theprogression of the pre-malignant cancer to a malignant form.

Cancers which can be treated by the methods of some embodiments of theinvention can be any solid or non-solid cancer and/or cancer metastasis.

According to specific embodiments, the cancer comprises malignantcancer.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);Burkitt lymphoma, Diffused large B cell lymphoma (DLBCL), smalllymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediategrade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); T cell lymphoma, Hodgkin lymphoma, chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Acutemyeloid leukemia (AML), Acute promyelocytic leukemia (APL), Hairy cellleukemia; chronic myeloblastic leukemia (CML); and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of breast cancer,colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, livercancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. The cancerous conditions amenablefor treatment of the invention include metastatic cancers.

According to specific embodiments, the cancer comprises pre-malignantcancer.

Pre-malignant cancers (or pre-cancers) are well characterized and knownin the art (refer, for example, to Berman J J. and Henson D E., 2003.Classifying the precancers: a metadata approach. BMC Med Inform DecisMak. 3:8). Classes of pre-malignant cancers amenable to treatment viathe method of the invention include acquired small or microscopicpre-malignant cancers, acquired large lesions with nuclear atypia,precursor lesions occurring with inherited hyperplastic syndromes thatprogress to cancer, and acquired diffuse hyperplasias and diffusemetaplasias. Examples of small or microscopic pre-malignant cancersinclude HGSIL (High grade squamous intraepithelial lesion of uterinecervix), AIN (anal intraepithelial neoplasia), dysplasia of vocal cord,aberrant crypts (of colon), PIN (prostatic intraepithelial neoplasia).

Examples of acquired large lesions with nuclear atypia include tubularadenoma, AILD (angioimmunoblastic lymphadenopathy with dysproteinemia),atypical meningioma, gastric polyp, large plaque parapsoriasis,myelodysplasia, papillary transitional cell carcinoma in-situ,refractory anemia with excess blasts, and Schneiderian papilloma.Examples of precursor lesions occurring with inherited hyperplasticsyndromes that progress to cancer include atypical mole syndrome, C celladenomatosis and MEA. Examples of acquired diffuse hyperplasias anddiffuse metaplasias include AIDS, atypical lymphoid hyperplasia, Paget'sdisease of bone, post-transplant lymphoproliferative disease andulcerative colitis.

In some embodiments of the invention, the diseases to be treated by afusion protein comprising SIRPα or the ECD thereof and 41BBL or ECDthereof, such as for example, SIRPα-G-41BBL are: Leukemia, Chronicmyelomonocytic leukemia (CMML), Chronic myelogenous leukemia (CML),Acute myeloid leukemia (AML), Non Hodgkin lymphoma (NHL), Diffuse LargeB Cell Lymphoma (DLBCL), B cell Chronic Lymphocytic Leukemia (B-CLL),Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Marginal ZoneLymphoma (MZL), Pre-B acute lymphoblastic leukemia (pre-B ALL),Leiomyosarcoma, Ovarian cancer, Breast cancer, Colon cancer, Bladdercancer, Glioblastoma, Hepatocellular carcinoma, Prostate cancer, Acutelymphoblastic leukemia (ALL), Multiple Myeloma, Non-small-cell lungcarcinoma (NSCLC), Colorectal cancer, Melanoma, Head and Neck Cancer,Marginal Zone B-cell Lymphoma, Pancreatic Ductal Adenocarcinoma, Braincancer

According to some embodiments of the invention the indications thediseases to be treated by a fusion protein comprising SIRPα or the ECDthereof and 41BBL or ECD thereof, such as for example, SIRPα-G-41BBLare: Acute myeloid leukemia, Bladder Cancer, Breast Cancer, chroniclymphocytic leukemia, Chronic myelogenous leukemia, Colorectal cancer,Diffuse large B-cell lymphoma, Epithelial Ovarian Cancer, EpithelialTumor, Fallopian Tube Cancer, Follicular Lymphoma, Glioblastomamultiform, Hepatocellular carcinoma, Head and Neck Cancer, Leukemia,Lymphoma, Mantle Cell Lymphoma, Melanoma, Mesothelioma, MultipleMyeloma, Nasopharyngeal Cancer, Non Hodgkin lymphoma, Non-small-celllung carcinoma, Ovarian Cancer, Prostate Cancer, Renal cell carcinoma.

According to specific embodiments, the cancer is selected from the groupconsisting of lymphoma, leukemia, colon cancer, pancreatic cancer,ovarian cancer, lung cancer and squamous cell carcinoma.

According to specific embodiments, the cancer is selected from the groupconsisting of lymphoma, carcinoma and leukemia.

According to specific embodiments, the cancer is colon carcinoma.

According to specific embodiments, the cancer is ovarian carcinoma.

According to specific embodiments, the cancer is lung carcinoma.

According to specific embodiments, the cancer is head and neckcarcinoma.

According to specific embodiments, the cancer is leukemia.

According to specific embodiments, the leukemia is selected from thegroup consisting of acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cellleukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, ( )ross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

According to specific embodiments, the leukemia is promyelocyticleukemia, acute myeloid leukemia or chronic myelogenous leukemia.

According to specific embodiments, the cancer is lymphoma.

According to specific embodiments, the lymphoma is B cell lymphoma

According to specific embodiments, the lymphoma is T cell lymphoma.

According to other specific embodiments, the lymphoma is Hodgkinslymphoma.

According to specific embodiments, the lymphoma is non-Hodgkinslymphoma.

According to specific embodiments, the non-Hodgkin's Lymphoma is aselected from the group consisting of aggressive NHL, transformed NHL,indolent NHL, relapsed NHL, refractory NHL, low grade non-Hodgkin'sLymphoma, follicular lymphoma, large cell lymphoma, B-cell lymphoma,T-cell lymphoma, Mantle cell lymphoma, Burkitt's lymphoma, NK celllymphoma, diffuse large B-cell lymphoma, acute lymphoblastic lymphoma,and cutaneous T cell cancer, including mycosos fungoides/Sezry syndrome.

According to specific embodiments, the cancer is multiple myeloma.

According to at least some embodiments, the multiple myeloma is selectedfrom the group consisting of multiple myeloma cancers which producelight chains of kappa-type and/or light chains of lambda-type;aggressive multiple myeloma, including primary plasma cell leukemia(PCL); benign plasma cell disorders such as MGUS (monoclonal gammopathyof undetermined significance), Waldenstrom's macroglobulinemia (WM, alsoknown as lymphoplasmacytic lymphoma) which may proceed to multiplemyeloma; smoldering multiple myeloma (SMM), indolent multiple myeloma,premalignant forms of multiple myeloma which may also proceed tomultiple myeloma; primary amyloidosis.

A Suggested Mode of Action of SIRPα-41BBL

In one embodiment of the invention, the chimera SIRPα-41BBL can be usedfor treating of cancer via the following possible mode-of-action:

-   -   Due to the relatively high expression of CD47 on the surface of        tumor cells and in the tumor micro-environment, the SIRPα moiety        of the SIRPα-41BBL chimera will target the molecule to tumor and        metastasis sites, and will bind the chimera to CD47 within the        tumor micro-environment.    -   Targeting the chimera to the tumor cells or/and tumor        micro-environment will facilitate an increase in SIRPα-41BBL        concertation in the tumor micro-environment and subsequent        oligomerization of the 4-1BBL moiety of the chimera at the tumor        site. Since oligomerization of 4-1BBL is a necessary step for        4-1BB signaling, this 4-1BBL binding and oligomerization will        deliver a 4-1BB co-stimulatory signal that will promote        activation of T-cells, B cells, NK cells, especially        Tumor-Infiltrating Lymphocytes (TILs), and other immune cells at        the tumor site, to kill cancer cells.    -   In addition to the 41BBL-41BB co-stimulatory signal, the binding        of the chimera's SIRPα moiety to CD47 in the tumor site will        compete with the endogenous SIRPα expressed on macrophages and        dendritic cells, thus, removing the inhibition on these cells        and further contributing to the phagocytosis of tumor cells and        to activation of dendritic cells and T cells in the tumor        micro-environment.

The above activities of SIRPA-41BBL are anticipated to lead to asynergistic effect on the activation of TILs, dendritic cells andmacrophages within the tumor micro-environment, which is expected to bemore specific and robust effect as compared to the effect of eachpeptide or ECD thereof separately, as well as when using the twodifferent peptides or ECD thereof in combination.

Thus, according to specific embodiments, the cancer is defined by thepresence of tumors that have tumor-infiltrating lymphocytes (TILs) inthe tumor micro-environment and/or tumors with expression of CD47 in thetumor micro-environment.

According to specific embodiments, the cancer is defined by the presenceof tumors that have tumor-infiltrating lymphocytes (TILs) in the tumormicro-environment and/or tumors with a relatively high expression ofCD47 in the tumor micro-environment.

According to specific embodiments, cells of the cancer express CD47.

According to specific embodiments, the disease comprises a diseaseassociated with immune suppression or immunosuppression caused bymedication (e.g. mTOR inhibitors, calcineurin inhibitor, steroids).

According to specific embodiments, the disease comprises HIV, Measles,influenza, LCCM, RSV, Human Rhinoviruses, EBV, CMV, Parvo viruses.

According to specific embodiments, the disease comprises an infection.

As used herein, the term “infection” of “infectious disease” refers to adisease induced by a pathogen. Specific examples of pathogens include,viral pathogens, bacterial pathogens e.g., intracellular mycobacterialpathogens (such as, for example, Mycobacterium tuberculosis),intracellular bacterial pathogens (such as, for example, Listeriamonocytogenes), or intracellular protozoan pathogens (such as, forexample, Leishmania and Trypanosoma).

Specific types of viral pathogens causing infectious diseases treatableaccording to the teachings of the present invention include, but are notlimited to, retroviruses, circoviruses, parvoviruses, papovaviruses,adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses,picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses,orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses,coronaviruses, arenaviruses, and filoviruses.

Specific examples of viral infections which may be treated according tothe teachings of the present invention include, but are not limited to,human immunodeficiency virus (HIV)-induced acquired immunodeficiencysyndrome (AIDS), influenza, rhinoviral infection, viral meningitis,Epstein-Barr virus (EBV) infection, hepatitis A, B or C virus infection,measles, papilloma virus infection/warts, cytomegalovirus (CMV)infection, Herpes simplex virus infection, yellow fever, Ebola virusinfection, rabies, etc.

According to specific embodiments, the compositions of the presentinvention (e.g. SIRPα-41BBL fusion protein, polynucleotide or nucleicacid construct encoding same and/or host-cell expressing same) can beadministered to a subject in combination with other established orexperimental therapeutic regimen to treat a disease that can benefitfrom activating immune cells (e.g. cancer) including, but not limited toanalgesics, chemotherapeutic agents, radiotherapeutic agents, cytotoxictherapies (conditioning), hormonal therapy, antibodies and othertreatment regimens (e.g., surgery) which are well known in the art.

According to specific embodiments, the compositions of the presentinvention (e.g. SIRPα-41BBL fusion protein, polynucleotide or nucleicacid construct encoding same and/or host-cell expressing same) can beadministered to a subject in combination with adoptive celltransplantation such as, but not limited to transplantation of bonemarrow cells, hematopoietic stem cells, PBMCs, cord blood stem cellsand/or induced pluripotent stem cells.

According to specific embodiments, the therapeutic agent administered incombination with the composition of the invention comprises ananti-cancer agent.

Thus, according to another aspect of the present invention, there isprovided a method of treating cancer comprising administering to asubject in need thereof an anti-cancer agent; and a SIRPα-41BBL fusionprotein, a polynucleotide encoding same, a nucleic acid constructencoding same or a host cell expressing same.

Anti-cancer agents that can be use with specific embodiments of theinvention include, but are not limited to the anti-cancer drugsAcivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin;Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium;Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; IdarubicinHydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; InterferonAlfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a;Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; LanreotideAcetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol;Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine;Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide;Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan;Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; TecogalanSodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide;Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa;Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate;Trestolone Acetate; Triciribine Phosphate; Trimetrexate; TrimetrexateGlucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard;Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; VincristineSulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; VinglycinateSulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; VinrosidineSulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;Zorubicin Hydrochloride. Additional antineoplastic agents include thosedisclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and BruceA. Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

According to specific embodiments, the anti-cancer agent comprises anantibody.

According to specific embodiments, the antibody is selected from thegroup consisting rituximab, cetuximab, trastuzumab, edrecolomab,alemtuzumab, gemtuzumab, ibritumomab, panitumumab, Belimumab,Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Blontuvetmab,Brentuximab vedotin, Catumaxomab, Cixutumumab, Daclizumab, Adalimumab,Bezlotoxumab, Certolizumab pegol, Citatuzumab bogatox, Daratumumab,Dinutuximab, Elotuzumab, Ertumaxomab, Etaracizumab, Gemtuzumabozogamicin, Girentuximab, Necitumumab, Obinutuzumab, Ofatumumab,Pertuzumab, Ramucirumab, Siltuximab, Tositumomab, Trastuzumab andipilimumab.

According to specific embodiments, the antibody is selected from thegroup consisting of rituximab and cetuximab.

According to specific embodiments, the therapeutic agent administered incombination with the composition of the invention comprises ananti-infection agent (e.g. antibiotics and anti-viral agents)

According to specific embodiments, the therapeutic agent administered incombination with the composition of the invention comprises an immunesuppressor agent (e.g. GCSF and other bone marrow stimulators, steroids)

According to specific embodiments the combination therapy has anadditive effect.

According to specific embodiments, the combination therapy has asynergistic effect.

According to another aspect of the present invention there is providedan article of manufacture identified for the treatment of a disease thatcan benefit from activating immune cells comprising a packaging materialpackaging a therapeutic agent for treating said disease; and aSIRPα-41BBL fusion protein, a polynucleotide encoding same, a nucleicacid construct encoding same or a host cell expressing same.

According to specific embodiments, the therapeutic agent for treatingsaid disease; and a SIRPα-41BBL fusion protein, a polynucleotideencoding same, a nucleic acid construct encoding same or a host cellexpressing same are packages in separate containers.

According to specific embodiments, the therapeutic agent for treatingsaid disease; and a SIRPα-41BBL fusion protein, a polynucleotide or anucleic acid encoding same, a nucleic acid construct encoding same or ahost cell expressing same are packages in a co-formulation.

As used herein, in one embodiment, the term “amino acid derivative” or“derivative” refers to a group derivable from a naturally ornon-naturally occurring amino acid, as described and exemplified herein.Amino acid derivatives are apparent to those of skill in the art andinclude, but are not limited to, ester, amino alcohol, amino aldehyde,amino lactone, and N-methyl derivatives of naturally and non-naturallyoccurring amino acids. In an embodiment, an amino acid derivative isprovided as a substituent of a compound described herein, wherein thesubstituent is —NH-G(Sc)-C(0)-Q or —OC(O)G(S_(c))-Q, wherein Q is —SR,—NRR or alkoxyl, R is hydrogen or alkyl, S_(c) is a side chain of anaturally occurring or non-naturally occurring amino acid and G is C₁-C₂alkyl. In certain embodiments, G is Ci alkyl and Sc is selected from thegroup consisting of hydrogen, alkyl, heteroalkyl, arylalkyl andheteroarylalkyl.

As used herein, in one embodiment, the term “peptide”, “polypeptide” or“protein” which are interchangeably used herein may be derived from anatural biological source, synthesized, or produced by recombinanttechnology. It may be generated in any manner known in the art ofpeptide or protein synthesis, including by chemical synthesis. For solidphase peptide synthesis, a summary of the many techniques may be foundin J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H.Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteinsand Peptides, vol. 2, p. 46, Academic Press (New York), 1973. Forclassical solution synthesis see G. Schroder and K. Lupke, The Peptides,vol. 1, Academic Press (New York), 1965. One or more of the amino acidsmay be modified, for example, by the addition of a chemical entity suchas a carbohydrate group, a phosphate group, a farnesyl group, anisofamesyt group, a fatty acid group, an acyl group (e.g., acetylgroup), a linker for conjugation, functionalization, or other knownprotecting/blocking groups. Modifications to the peptide or protein canbe introduced by gene synthesis, site-directed (e.g., PCR based) orrandom mutagenesis (e.g., EMS) by exonuclease deletion, by chemicalmodification, or by fusion of polynucleotide sequences encoding aheterologous domain or binding protein, for example.

As used herein, in one embodiment, the term “peptide,” may be fragments,derivatives, analogs, or variants of the foregoing peptides, and anycombination thereof. Fragments of peptides, as that term or phrase isused herein, include proteolytic fragments, as well as deletionfragments. Variants of peptides include fragments and peptides withaltered amino acid sequences due to amino acid substitutions, deletions,or insertions.

Variants may occur naturally or be non-naturally occurring. Examplesinclude fusion proteins, peptides having one or more residues chemicallyderivatized by reaction of a functional side group, and peptides thatcontain one or more naturally occurring amino acid derivatives of thetwenty standard amino acids. These modifications may also include theincorporation of D-amino acids, or other non-encoded amino-acids. In oneembodiment, none of the modifications should substantially interferewith the desired biological activity of the peptide, fragment thereof.In another embodiment, modifications may alter a characteristic of thepeptide, fragment thereof, for instance stability or half-life, withoutinterfering with the desired biological activity of the peptide,fragment thereof. In one embodiment, as used herein the terms “peptide”and “protein” may be used interchangeably having all the same meaningsand qualities.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the peptide of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the peptide can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the peptide and the cleavable moiety and the peptide can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

In one embodiment, each of the peptides that forms the fusion protein(also termed here “the peptide”) of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In one embodiment, production of a peptide of this invention is usingrecombinant DNA technology. A “recombinant” peptide, or protein refersto a peptide, or protein produced by recombinant DNA techniques; i.e.,produced from cells transformed by an exogenous DNA construct encodingthe desired peptide or protein.

Thus, according to another aspect of the present invention, there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding any of the above described fusion proteins.

According to specific embodiments, the polynucleotide comprises SEQ IDNO: 8.

According to specific embodiments, the polynucleotide consists of SEQ IDNO: 8.

According to specific embodiments, the polynucleotide is least about70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to thenucleic sequence as set forth in SEQ ID No. 8.

As used herein the term “polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above).

To express exogenous SIRPα-41BBL in mammalian cells, a polynucleotidesequence encoding SIRPα-41BBL is preferably ligated into a nucleic acidconstruct suitable for mammalian cell expression. Such a nucleic acidconstruct includes a promoter sequence for directing transcription ofthe polynucleotide sequence in the cell in a constitutive or induciblemanner.

Hence, according to specific embodiments, there is provided nucleic acidconstruct comprising the polynucleotide and a regulatory element fordirecting expression of said polynucleotide in a host cell.

The nucleic acid construct (also referred to herein as an “expressionvector”) of some embodiments of the invention includes additionalsequences which render this vector suitable for replication andintegration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). In addition, a typical cloning vectors may alsocontain a transcription and translation initiation sequence,transcription and translation terminator and a polyadenylation signal.By way of example, such constructs will typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof.

The nucleic acid construct of some embodiments of the inventiontypically includes a signal sequence for secretion of the peptide from ahost cell in which it is placed. Preferably the signal sequence for thispurpose is a mammalian signal sequence or the signal sequence of thepolypeptide variants of some embodiments of the invention.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of someembodiments of the invention is active in the specific cell populationtransformed. Examples of cell type-specific and/or tissue-specificpromoters include promoters such as albumin that is liver specific[Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specificpromoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; inparticular promoters of T-cell receptors [Winoto et al., (1989) EMBO J.8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740],neuron-specific promoters such as the neurofilament promoter [Byrne etal. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specificpromoters [Edlunch et al. (1985) Science 230:912-916] or mammarygland-specific promoters such as the milk whey promoter (U.S. Pat. No.4,873,316 and European Application Publication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for some embodiments of the inventioninclude those derived from polyoma virus, human or murinecytomegalovirus (CMV), the long term repeat from various retrovirusessuch as murine leukemia virus, murine or Rous sarcoma virus and HIV.See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of SIRPα-41BBL mRNA translation. Twodistinct sequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for some embodiments of theinvention include those derived from SV40.

In addition to the elements already described, the expression vector ofsome embodiments of the invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote the extra chromosomal replication ofthe viral genome in permissive cell types.

Plasmids bearing these viral replicons are replicated episomally as longas the appropriate factors are provided by genes either carried on theplasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of some embodiments of the invention can furtherinclude additional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

It will be appreciated that the individual elements comprised in theexpression vector can be arranged in a variety of configurations. Forexample, enhancer elements, promoters and the like, and even thepolynucleotide sequence(s) encoding a SIRPα-41BBL can be arranged in a“head-to-tail” configuration, may be present as an inverted complement,or in a complementary configuration, as an anti-parallel strand. Whilesuch variety of configuration is more likely to occur with non-codingelements of the expression vector, alternative configurations of thecoding sequence within the expression vector are also envisioned.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby some embodiments of the invention will depend on the cell typetransformed. The ability to select suitable vectors according to thecell type transformed is well within the capabilities of the ordinaryskilled artisan and as such no general description of selectionconsideration is provided herein. For example, bone marrow cells can betargeted using the human T cell leukemia virus type I (HTLV-I) andkidney cells may be targeted using the heterologous promoter present inthe baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) asdescribed in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression ofSIRPα-41BBL since they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the life cycleof, for example, retrovirus and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. This is in contrast to vertical-type of infection in whichthe infectious agent spreads only through daughter progeny. Viralvectors can also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

Various methods can be used to introduce the expression vector of someembodiments of the invention into cells. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. In addition, such a construct typicallyincludes a signal sequence for secretion of the peptide from a host cellin which it is placed. Preferably the signal sequence for this purposeis a mammalian signal sequence or the signal sequence of the polypeptidevariants of some embodiments of the invention. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction sites and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. Other vectors can be usedthat are non-viral, such as cationic lipids, polylysine, and dendrimers.

As mentioned, other than containing the necessary elements for thetranscription and translation of the inserted coding sequence, theexpression construct of some embodiments of the invention can alsoinclude sequences engineered to enhance stability, production,purification, yield or toxicity of the expressed peptide. For example,the expression of a fusion protein or a cleavable fusion proteincomprising the SIRPα-41BBL protein of some embodiments of the inventionand a heterologous protein can be engineered. Such a fusion protein canbe designed so that the fusion protein can be readily isolated byaffinity chromatography; e.g., by immobilization on a column specificfor the heterologous protein. Where a cleavage site is engineeredbetween the SIRPα-41BBL protein and the heterologous protein, theSIRPα-41BBL protein can be released from the chromatographic column bytreatment with an appropriate enzyme or agent that disrupts the cleavagesite [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; andGardella et al., (1990) J. Biol. Chem. 265:15854-15859].

The present invention also contemplates cells comprising the compositiondescribed herein.

Thus, according to specific embodiments, there is provided a host cellcomprising the SIRPα-41BBL fusion protein, the polynucleotide encodingsame or the nucleic acid construct encoding same.

As mentioned hereinabove, a variety of prokaryotic or eukaryotic cellscan be used as host-expression systems to express the polypeptides ofsome embodiments of the invention. These include, but are not limitedto, microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the coding sequence; yeast transformed with recombinant yeastexpression vectors containing the coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors, such as Ti plasmid, containingthe coding sequence. Mammalian expression systems can also be used toexpress the polypeptides of some embodiments of the invention.

Examples of bacterial constructs include the pET series of E. coliexpression vectors (Studier et al. (1990) Methods in Enzymol.185:60-89).

Examples of eukaryotic cells which may be used along with the teachingsof the invention include but are not limited to, mammalian cells, fungalcells, yeast cells, insect cells, algal cells or plant cells.

In yeast, a number of vectors containing constitutive or induciblepromoters can be used, as disclosed in U.S. Pat. No. 5,932,447.Alternatively, vectors can be used which promote integration of foreignDNA sequences into the yeast chromosome.

In cases where plant expression vectors are used, the expression of thecoding sequence can be driven by a number of promoters. For example,viral promoters such as the 35S RNA and 19S RNA promoters of CaMV[Brisson et al. (1984) Nature 310:511-514], or the coat protein promoterto TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used.Alternatively, plant promoters such as the small subunit of RUBISCO[Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984)Science 224:838-843] or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B [Gurley et al. (1986) Mol. Cell. Biol. 6:559-565] can be used.These constructs can be introduced into plant cells using Ti plasmid, Riplasmid, plant viral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.

Other expression systems such as insects and mammalian host cell systemswhich are well known in the art can also be used by some embodiments ofthe invention.

According to specific embodiments the cell is a mammalian cell.

According to specific embodiment, the cell is a human cell.

According to a specific embodiment, the cell is a cell line.

According to another specific embodiment, the cell is a primary cell.

The cell may be derived from a suitable tissue including but not limitedto blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen,thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair,skin, bone, breast, uterus, bladder, spinal cord, or various kinds ofbody fluids. The cells may be derived from any developmental stageincluding embryo, fetal and adult stages, as well as developmentalorigin i.e., ectodermal, mesodermal, and endodermal origin.

Non limiting examples of mammalian cells include monkey kidney CV1 linetransformed by SV40 (COS, e.g. COS-7, ATCC CRL 1651); human embryonickidney line (HEK293 or HEK293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 1977); baby hamster kidneycells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 1980); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HeLa, ATCC CCL 2); NIH3T3, Jurkat, caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y. Acad. Sci., 383:44-68 1982); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2), PER.C6, K562, and Chinesehamster ovary cells (CHO).

According to some embodiments of the invention, the mammalian cell isselected from the group consisting of a Chinese Hamster Ovary (CHO),HEK293, PER.C6, HT1080, NSO, Sp2/0, BHK, Namalwa, COS, HeLa and Verocell.

According to some embodiments of the invention, the host cell comprisesa Chinese Hamster Ovary (CHO), PER.C6 and 293 (e.g., Expi293F) cell.

According to another aspect of the present invention, there is provideda method of producing a SIRPα-41BBL fusion protein, the methodcomprising expressing in a host cell the polynucleotide or the nucleicacid construct described herein.

According to specific embodiments, the methods comprising isolating thefusion protein.

According to specific embodiments, recovery of the recombinantpolypeptide is effected following an appropriate time in culture. Thephrase “recovering the recombinant polypeptide” refers to collecting thewhole fermentation medium containing the polypeptide and need not implyadditional steps of separation or purification. Notwithstanding theabove, polypeptides of some embodiments of the invention can be purifiedusing a variety of standard protein purification techniques, such as,but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, mix mode chromatography,metal affinity chromatography, Lectins affinity chromatography,chromatofocusing and differential solubilization.

In some embodiments, the recombinant peptides, fragments thereof orpeptides are synthesized and purified; their therapeutic efficacy can beassayed either in vivo or in vitro. In one embodiment, the activities ofthe recombinant fragments or peptides of the present invention can beascertained using various assays including cell viability, survival oftransgenic mice, and expression of megakaryocytic and lymphoid RNAmarkers.

In one embodiment, a peptide of this invention comprises at least 3amino acids. In another embodiment, a peptide comprises at least 5 aminoacids. In another embodiment, a peptide comprises at least 10 aminoacids. In another embodiment, a peptide comprises at least 20 aminoacids. In another embodiment, a peptide comprises at least 25 aminoacids. In other embodiments, a peptide comprises at least 30 amino acidsor at least 50 amino acids or 75 amino acids, or 100 amino acids, or 125amino acids, or 150 amino acids, or 200 amino acids, or 250 amino acidsor 300 amino acids or 350 amino acids or 400 amino acids. In oneembodiment, a peptide of this invention consists essentially of at least5 amino acids. In another embodiment, a peptide consists essentially ofat least 10 amino acids. In other embodiments, a peptide consistsessentially of at least 30 amino acids or at least 50 amino acids or 75amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids,or 200 amino acids, or 250 amino acids or 300 amino acids or 350 aminoacids or 400 amino acids. In one embodiment, a peptide of this inventionconsists of at least 5 amino acids. In another embodiment, a peptideconsists of at least 10 amino acids. In other embodiments, a peptideconsists of at least 30 amino acids or at least 50 amino acids or 75amino acids, or 100 amino acids, or 125 amino acids, or 150 amino acids,or 200 amino acids, or 250 amino acids or 300 amino acids or 350 aminoacids or 400 amino acids or 500 or 600 or 700 amino acids.

As used herein, in one embodiment, the terms “peptide” and “fragment”may be used interchangeably having all the same meanings and qualities.As used herein in, in one embodiment the term “peptide” includes nativepeptides (either degradation products, synthetically synthesizedpeptides or recombinant peptides) and peptidomimetics (typically,synthetically synthesized peptides), such as peptoids and semipeptoidswhich are peptide analogs, which may have, for example, modificationsrendering the peptides more stable while in a body or more capable ofpenetrating into bacterial cells. Such modifications include, but arenot limited to N terminus modification, C terminus modification, peptidebond modification, including, but not limited to, CH2-NH, CH2-S,CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbonemodifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further details in this respectare provided herein under.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylamine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

In one embodiment, the peptide of this invention further comprises adetectable tag. As used herein, in one embodiment the term “detectabletag” refers to any moiety that can be detected by a skilled practitionerusing art known techniques. Detectable tags for use in the screeningmethods of the present invention may be peptide sequences. Optionallythe detectable tag may be removable by chemical agents or by enzymaticmeans, such as proteolysis. For example the term “detectable tag”includes chitin binding protein (CBP)-tag, maltose binding protein(MBP)-tag, glutathione-S-transferase (GST)-tag, poly(His)-tag, FLAG tag,Epitope tags, such as, V5-tag, c-myc-tag, and HA-tag, and fluorescencetags such as green fluorescent protein (GFP), red fluorescent protein(RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP),and cyan fluorescent protein (CFP); as well as derivatives of thesetags, or any tag known in the art. The term “detectable tag” alsoincludes the term “detectable marker”.

In one embodiment, a peptide of this invention is an isolated peptide.Such an isolated peptide may include a peptide-tag.

The peptides of some embodiments of the invention are preferablyutilized in a linear form, although it will be appreciated that in caseswhere cyclicization does not severely interfere with peptidecharacteristics, cyclic forms of the peptide can also be utilized.

As used herein, in one embodiment the term “amino acid” refers tonaturally occurring and synthetic α, β γ or δ amino acids, and includesbut is not limited to, amino acids found in proteins, i.e. glycine,alanine, valine, leucine, isoleucine, methionine, phenylalanine,tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartate, glutamate, lysine, arginine and histidine. Incertain embodiments, the amino acid is in the L-configuration.Alternatively, the amino acid can be a derivative of alanyl, valinyl,leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl.

Since the present peptides are preferably utilized in therapeutics ordiagnostics which require the peptides to be in soluble form, thepeptides of some embodiments of the invention preferably include one ormore non-natural or natural polar amino acids, including but not limitedto serine and threonine which are capable of increasing peptidesolubility due to their hydroxyl-containing side chain.

As used herein, in one embodiment the phrase “Conservatively modifiedvariants” applies to both amino acid and nucleic acid sequences. “Aminoacid variants” refers to amino acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical or associated(e.g., naturally contiguous) sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode most proteins. For instance, the codons GCA, GCC, GCG and GCU allencode the amino acid alanine. Thus, at every position where an alanineis specified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations”, which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, silent variations of a nucleic acidwhich encodes a polypeptide is implicit in a described sequence withrespect to the expression product.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant”, including where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Guidance concerning whichamino acid changes are likely to be phenotypically silent can also befound in Bowie et al., 1990, Science 247: 1306 1310. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles. Typical conservativesubstitutions include but are not limited to: 1) Alanine (A), Glycine(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). Amino acids canbe substituted based upon properties associated with side chains, forexample, amino acids with polar side chains may be substituted, forexample, Serine (S) and Threonine (T); amino acids based on theelectrical charge of a side chains, for example, Arginine (R) andHistidine (H); and amino acids that have hydrophobic side chains, forexample, Valine (V) and Leucine (L). As indicated, changes are typicallyof a minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein.

Protein Chemical Modifications

In the present invention any part of a protein of the invention mayoptionally be chemically modified, i.e. changed by addition offunctional groups. For example the side amino acid residues appearing inthe native sequence may optionally be modified, although as describedbelow alternatively other parts of the protein may optionally bemodified, in addition to or in place of the side amino acid residues.The modification may optionally be performed during synthesis of themolecule if a chemical synthetic process is followed, for example byadding a chemically modified amino acid. However, chemical modificationof an amino acid when it is already present in the molecule (“in situ”modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification”, when referring to aprotein or peptide according to the present invention, refers to aprotein or peptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Examples of the numerous knownmodifications typically include, but are not limited to: acetylation,acylation, amidation, ADP-ribosylation, glycosylation, GPI anchorformation, covalent attachment of a lipid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiquitination, or any similar process.

Other types of modifications optionally include the addition of acycloalkane moiety to a biological molecule, such as a protein, asdescribed in PCT Application No. WO 2006/050262, hereby incorporated byreference as if fully set forth herein. These moieties are designed foruse with biomolecules and may optionally be used to impart variousproperties to proteins.

Furthermore, optionally any point on a protein may be modified. Forexample, pegylation of a glycosylation moiety on a protein mayoptionally be performed, as described in PCT Application No. WO2006/050247, hereby incorporated by reference as if fully set forthherein. One or more polyethylene glycol (PEG) groups may optionally beadded to O-linked and/or N-linked glycosylation. The PEG group mayoptionally be branched or linear. Optionally any type of water-solublepolymer may be attached to a glycosylation site on a protein through aglycosyl linker.

By “PEGylated protein” is meant a protein, or a fragment thereof havingbiological activity, having a polyethylene glycol (PEG) moietycovalently bound to an amino acid residue of the protein.

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., with thiol,triflate, tresylate, azirdine, oxirane, or preferably with a maleimidemoiety). Compounds such as maleimido monomethoxy PEG are exemplary oractivated PEG compounds of the invention. Other polyalkylene glycolcompounds, such as polypropylene glycol, may be used in the presentinvention. Other appropriate polyalkylene glycol compounds include, butare not limited to, charged or neutral polymers of the following types:dextran, colominic acids or other carbohydrate based polymers, polymersof amino acids, and biotin derivatives.

Altered Glycosylation Protein Modification

Proteins of the invention may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties deleted, and/or having at least oneglycosylation site added to the original protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins of the invention isconveniently accomplished by altering the amino acid sequence of theprotein such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues in the sequence of the original protein(for O-linked glycosylation sites). The protein's amino acid sequencemay also be altered by introducing changes at the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. Depending on the coupling mode used, thesugars may be attached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulfhydryl groups such as those of cysteine, (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline,(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev.Biochem., 22: 259-306 (1981).

Removal of any carbohydrate moieties present on proteins of theinvention may be accomplished chemically, enzymatically or byintroducing changes at the DNA level. Chemical deglycosylation requiresexposure of the protein to trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), leaving the amino acid sequence intact.

Chemical deglycosylation is described by Hakimuddin et al., Arch.Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

Pharmaceutical Compositions

The compositions (e.g. SRIPα-41BBL fusion protein, polynucleotideencoding same, nucleic acid construct encoding same and/or cells) ofsome embodiments of the invention can be administered to an organism perse, or in a pharmaceutical composition where it is mixed with suitablecarriers or excipients.

The present invention, in some embodiments, features a pharmaceuticalcomposition comprising a therapeutically effective amount of atherapeutic agent according to the present invention. According to thepresent invention the therapeutic agent could be a polypeptide asdescribed herein. The pharmaceutical composition according to thepresent invention is further used for the treatment of cancer or animmune related disorder as described herein. The therapeutic agents ofthe present invention can be provided to the subject alone, or as partof a pharmaceutical composition where they are mixed with apharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the composition (e.g.SIRPα-41BBL fusion protein, polynucleotide, nucleic acid constructand/or cells described herein) accountable for the biological effect.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., a polypeptide, apolynucleotide, a nucleic acid construct and/or cell as describedherein, may include one or more pharmaceutically acceptable salts. A“pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition according to at least some embodiments ofthe present invention also may include a pharmaceutically acceptableanti-oxidants. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Apharmaceutical composition according to at least some embodiments of thepresent invention also may include additives such as detergents andsolubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80(polysorbate-80)) and preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol).

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions according to at least someembodiments of the present invention include water, buffered saline ofvarious buffer content (e.g., Tris-HCl, acetate, phosphate), pH andionic strength, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate.

Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the present invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for therapeutic agentsaccording to at least some embodiments of the present invention includeintravascular delivery (e.g. injection or infusion), intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral,enteral, rectal, pulmonary (e.g. inhalation), nasal, topical (includingtransdermal, buccal and sublingual), intravesical, intravitreal,intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular,intra-cerebral, and convection enhanced diffusion), CNS delivery (e.g.intrathecal, perispinal, and intra-spinal) or parenteral (includingsubcutaneous, intramuscular, intraperitoneal, intravenous (IV) andintradermal), transdermal (either passively or using iontophoresis orelectroporation), transmucosal (e.g., sublingual administration, nasal,vaginal, rectal, or sublingual), administration or administration via animplant, or other parenteral routes of administration, for example byinjection or infusion, or other delivery routes and/or forms ofadministration known in the art. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion or using bioerodible inserts, and can be formulated in dosageforms appropriate for each route of administration. In a specificembodiment, a protein, a therapeutic agent or a pharmaceuticalcomposition according to at least some embodiments of the presentinvention can be administered intraperitoneally or intravenously.

Compositions of the present invention can be delivered to the lungswhile inhaling and traverse across the lung epithelial lining to theblood stream when delivered either as an aerosol or spray driedparticles having an aerodynamic diameter of less than about 5 microns. Awide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder preparations approved or in clinical trialswhere the technology could be applied to the formulations describedherein.

In some in vivo approaches, the compositions disclosed herein areadministered to a subject in a therapeutically effective amount. As usedherein the term “effective amount” or “therapeutically effective amount”means a dosage sufficient to treat, inhibit, or alleviate one or moresymptoms of the disorder being treated or to otherwise provide a desiredpharmacologic and/or physiologic effect. The precise dosage will varyaccording to a variety of factors such as subject-dependent variables(e.g., age, immune system health, etc.), the disease, and the treatmentbeing effected. For the polypeptide compositions disclosed herein, thepolynucleotides and nucleic acids constructs encoding the same and thecells described herein, as further studies are conducted, informationwill emerge regarding appropriate dosage levels for treatment of variousconditions in various patients, and the ordinary skilled worker,considering the therapeutic context, age, and general health of therecipient, will be able to ascertain proper dosing. The selected dosagedepends upon the desired therapeutic effect, on the route ofadministration, and on the duration of the treatment desired. Forpolypeptide compositions, generally dosage levels of 0.0001 to 100 mg/kgof body weight daily are administered to mammals and more usually 0.001to 20 mg/kg. For example dosages can be 0.3 mg/kg body weight, 1 mg/kgbody weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration 5 times per week, 4 times per week, 3 times perweek, 2 times per week, once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months.

Generally, for intravenous injection or infusion, dosage may be lower.Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of thepresent invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

Optionally the polypeptide formulation may be administered in an amountbetween 0.0001 to 100 mg/kg weight of the patient/day, preferablybetween 0.001 to 20.0 mg/kg/day, according to any suitable timingregimen. A therapeutic composition according to at least someembodiments according to at least some embodiments of the presentinvention can be administered, for example, three times a day, twice aday, once a day, three times weekly, twice weekly or once weekly, onceevery two weeks or 3, 4, 5, 6, 7 or 8 weeks. Moreover, the compositioncan be administered over a short or long period of time (e.g., 1 week, 1month, 1 year, 5 years).

Alternatively, therapeutic agent such as the compositions disclosedherein can be administered as a sustained release formulation, in whichcase less frequent administration is required. Dosage and frequency varydepending on the half-life of the therapeutic agent in the patient. Ingeneral, human antibodies show the longest half-life, followed byhumanized antibodies, chimeric antibodies, and nonhuman antibodies. Thehalf-life for fusion proteins may vary widely. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

A “therapeutically effective dosage” of a polypeptide as disclosedherein preferably results in a decrease in severity of disease symptoms,an increase in frequency and duration of disease symptom-free periods,an increase in lifespan, disease remission, or a prevention or reductionof impairment or disability due to the disease affliction.

One of ordinary skill in the art would be able to determine atherapeutically effective amount, especially in light of the detaileddisclosure provided herein, based on such factors as the subject's size,the severity of the subject's symptoms, and the particular compositionor route of administration selected.

In certain embodiments, the polypeptide, polynucleotide, nucleic acidconstruct or cells compositions are administered locally, for example byinjection directly into a site to be treated.

Typically, the injection causes an increased localized concentration ofthe polypeptide, polynucleotide, nucleic acid construct or cellscompositions which is greater than that which can be achieved bysystemic administration. The polypeptide compositions can be combinedwith a matrix as described above to assist in creating an increasedlocalized concentration of the polypeptide compositions by reducing thepassive diffusion of the polypeptides out of the site to be treated.

Pharmaceutical compositions of the present invention may be administeredwith medical devices known in the art. For example, in an optionalembodiment, a pharmaceutical composition according to at least someembodiments of the present invention can be administered with a needleshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in an optional embodiment, a therapeuticcomposition according to at least some embodiments of the presentinvention can be administered with a needles hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.Examples of well-known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art. Incertain embodiments, to ensure that the therapeutic compounds accordingto at least some embodiments of the present invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134); p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273.

Formulations for Parenteral Administration

In a further embodiment, compositions disclosed herein, including thosecontaining peptides and polypeptides, are administered in an aqueoussolution, by parenteral injection. The formulation may also be in theform of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of a peptide orpolypeptide, polynucleotide, nucleic acid construct or cells describedherein, and optionally include pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions optionally include one or more for the following:diluents, sterile water, buffered saline of various buffer content(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; andadditives such as detergents and solubilizing agents (e.g., TWEEN 20(polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., watersoluble antioxidants such as ascorbic acid, sodium metabisulfite,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite; oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol; and metal chelating agents, such as citricacid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid), and preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are ethanol, propylene glycol,polyethylene glycol, vegetable oils, such as olive oil and corn oil,gelatin, and injectable organic esters such as ethyl oleate. Theformulations may be freeze dried (lyophilized) or vacuum dried andredissolved/resuspended immediately before use. The formulation may besterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

Formulations for Topical Administration

Various compositions (e.g., polypeptides) disclosed herein can beapplied topically. Topical administration does not work well for mostpeptide formulations, although it can be effective especially if appliedto the lungs, nasal, oral (sublingual, buccal), vaginal, or rectalmucosa.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder preparations approved or in clinical trialswhere the technology could be applied to the formulations describedherein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations will require theinclusion of penetration enhancers.

Controlled Delivery Polymeric Matrices

Various compositions (e.g., polypeptides) disclosed herein may also beadministered in controlled release formulations. Controlled releasepolymeric devices can be made for long term release systemicallyfollowing implantation of a polymeric device (rod, cylinder, film, disk)or injection (microparticles). The matrix can be in the form ofmicroparticles such as microspheres, where peptides are dispersed withina solid polymeric matrix or microcapsules, where the core is of adifferent material than the polymeric shell, and the peptide isdispersed or suspended in the core, which may be liquid or solid innature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of polypeptides or nucleic acids encoding the polypeptides,although biodegradable matrices are preferred. These may be natural orsynthetic polymers, although synthetic polymers are preferred due to thebetter characterization of degradation and release profiles. The polymeris selected based on the period over which release is desired. In somecases linear release may be most useful, although in others a pulserelease or “bulk release” may provide more effective results. Thepolymer may be in the form of a hydrogel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl Polymer ScL, 35:755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection—which will typically deliver a dosage that ismuch less than the dosage for treatment of an entire body—or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

EXAMPLES Proof of Concept (POC) Experiments Manufacturing of aHis-Tagged SIRPα-41BBL

For initial POC analysis, a histidine-tagged protein is produced. A cDNAsequence, coding for a 6-His-tagged SIRPα-41BBL, is sub-cloned into amammalian expression vector. Transfection-grade plasmid preparation isused for plasmid transfection into Expi293 cells or other cell-lines.The supernatant of the Expi293 expressing cells (100 ml scale) isassessed for SIRPα-41BBL production by reduced and non-reduced SDS-PAGEand Western blot (WB) with an anti-His antibody. His-tagged SIRPα-41BBLis then purified from a positive supernatant by one-step affinity basedpurification (Nickel beads). The production of the tagged chimeraprotein is verified by SDS-PAGE and Western blot analysis using specificantibodies against each domain of the molecule (i.e. the extracellulardomain each of SIRPα and 41BBL).

Experiment 1A—Production of a His-Tagged SIRPα-41BBL Fusion Protein

Production of His-tag SIRPα-41BBL fusion protein (SEQ ID NO: 5) waseffected in Expi293F cells transfected by a pcDNA3.4 expression vectorcloned with coding sequence for the full fusion protein. The sequencewas cloned into the vector using EcoRI and HindIII restriction enzymes,with addition of Kozak sequence, artificial signal peptide and 6 His-tagin the N terminus and a stop codon in the C terminus (SEQ ID NO: 15).

The protein was collected from the supernatant of cell culture, andpurified by one-step purification by HisTrap™ FF Crude column.

Experiment 1B—the Produced SIRPα-41BBL Fusion Protein Contains BothDomains

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove, Protein marker: Spectra BR (Thermo FisherScientific, cat#26634), anti-SIRPα (SHPS1) (Cell Signaling, cat#13379),anti-41BB-L (BioVision, 5369-100), mouse-anti-His mAb (GenScript,Cat.No. A00186), secondary Goat Anti-Rabbit IgG (H+L)-HRP Conjugate(1:3333) (R&D, cat#170-6515), Recombinant hSIRPα 0.1 mg/ml (4546-SA-050)R&D, Recombinant h41BB-L (TNFSF9) 0.1 mg/ml (8460 LF) Cell SignalingStripping buffer (Thermoscientific, cat#21059), Protein De-glycosylationMix: (NEB p6044).

Methods—

Proteins (250 ng per lane) were treated at denaturing or non denaturingconditions (in sample buffer containing P3-mercaptoethanol and boiledfor 5 minutes at 95° C., or, in sample buffer without P3-mercaptoethanolwithout heating, respectively) and separated on 12% SDS-PAGE gel,followed by Western blotting. De-glycosylation treatment was effected byPNGase F enzyme according to the Protein De-glycosylation Mixmanufacturer instructions.

Results—

Western blot analysis of His-tagged SIRPα-41BBL (SEQ ID NO: 5) separatedon a SDS-PAGE under denaturing conditions followed by immunoblottingwith an anti His-tag antibody (FIG. 1) or an anti-41BBL antibody (FIG.2A) demonstrated that both the N-terminal side of the molecule and theC-terminal side of the molecule are present. Although the predictedmolecular weight of the protein according to its amino acid sequence isapproximately 60 kDa, the protein migrated in denaturing conditions asapproximately the size of 85 kDa. This shift was found to be related tothe glycosylation of the protein, as determined by treating the proteinwith PNGase F enzyme that removes almost all N-linked oligosaccharidesfrom glycoproteins. Following the treatment, a major band of around 60kDa was observed (FIG. 2C).

When separated on a SDS-PAGE under non-denaturing conditions (FIGS. 1and 2B) the His-tagged SIRPα-41BBL (SEQ ID NO: 5) was detected at thesame molecular weight, as in the denaturing conditions (FIGS. 1, 2A and2B). Additional bands of higher molecular weight were also detected,which were stronger under the non denaturing conditions compared to thedenaturing conditions. This might suggest a formation of a multimer,probably a trimer according to the size of the multimer and the factthat 41BBL protein naturally tends to form trimers (Eun-Young et al,2010, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 12, pp.9202-9210).

Experiment 1C—Binding Analysis of the SIRPα and 4-1BBL Moieties of theChimera to CD47 and 41BB

The binding of the SIRPα domain of the molecule to CD47 and the bindingof the 41BBL domain of the molecule to 41BB was determined by thebio-layer interferometry Blitz® assay.

Materials—

CD47:FC (Sino Biological, cat #12283-HO2H), 41BB:FC (Sino Biological,cat #10041-HO3H), His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) producedas described in Experiment 1A hereinabove; PD1-CD70 protein (SEQ ID NO:6, as a negative control).

Methods and Results—

The biosensor was pre-loaded with CD47:Fc, which led to a stableassociation plateau (FIG. 3A). Upon subsequent incubation withhis-tagged SIRPα-41BBL (SEQ ID NO: 5) a rapid association of thehis-tagged SIRPα-41BBL to CD47:Fc was detected (FIG. 3A). Similarincubation with control protein PD1-CD70 (composed of a PD-1 domainfused to CD70, SEQ ID NO: 6), did not lead to any binding to CD47:Fc(FIG. 3A). Furthermore, when the biosensor was not pre-loaded withCD47:Fc, the his-tagged SIRPα-41BBL did not associate (FIG. 3A, bottomline). Upon reaching a stable association plateau, the biosensor waswashed with medium to determine the off-rate of the his-taggedSIRPα-41BBL from CD47:Fc. The dissociation of the his-tagged SIRPα-41BBLfrom the CD47:Fc-loaded biosensor was very slow, suggesting stableinteraction of SIRPα with CD47.

Upon similar loading of the biosensor with 41BB:Fc, binding of the 41BBLunit of his-tagged SIRPα-41BBL (SEQ ID NO: 5) was evaluated (FIG. 3B).As with the SIRPα domain, the 41BBL domain of the his-tagged SIRPα-41BBLrapidly bound to its target receptor (FIG. 3B), with the off-rate forthe 41BBL/41BB interaction being also very slow, as evident from thelimited dissociation occurring during the last dissociation phase.Control treatment with a PD1-CD70 (SEQ ID NO: 6), lacking the 41BBLdomain, did not result in any detectable binding to 41BBL:Fc (FIG. 3B).Further, in the absence of pre-loading with 41BB:Fc, His-taggedSIRPα-41BBL did not detectably bind to the biosensor (FIG. 3B, bottomline).

Taken together, both domains of His-tagged SIRPα-41BBL (SEQ ID NO: 5)retain functional binding activity for their cognate receptors.

Experiment 1D—Binding Analysis of the SIRPα and 41BBL Moieties of theChimera to CD47

The binding of the SIRPα domain of the molecule to human CD47 isevaluated by using HT1080 cells or CHO-K1 cell or another cell lineoverexpressing CD47 or with a cancer cell line that is known to expressCD47 at high levels. CD47 knock-out cells are serving as negativecontrol. Cells are stained with different concentrations of His-taggedSIRPα-41BBL, and then by a secondary anti 41BBL antibody. Binding isanalyzed by flow cytometry using fluorescence-activated cell sorting(FACS). The use of different concentrations of the chimera allows todetermine the affinity of the molecule to the CD47. In this bindingtest, a recombinant SIRPα is also used as competitor to the SIRPα-41BBLin order to verify the specificity of the binding. Antibodies that blockthe interaction between SIRPα and CD47 can be used as well for the samepurpose.

The binding of the 41BBL moiety of the chimera to human 41BB is testedby using HT1080 cells or another cell line that are overexpressing 41BB.Cells are stained with different concentrations of SIRPα-41BBL and thenby a secondary anti SIRPα antibody, and binding affinity is analyzed byFACS. In this binding test, a recombinant 41BBL is used as a competitorto the SIRPα-41BBL in order to verify the specificity of the binding.Antibodies that block the interaction between 41BB and 41BBL can be usedfor the same purpose as well.

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove; CHO-WT and CHO-CD47 cell lines (Bommel et al,2017), Fixable Viability Dye (BD Biosciences, cat#562247), Human Fcblocker, True stain FCX (Biolegend, cat#422302), and the followingantibodies:

Target Fluor Cat# Manufacturer Antibodies used anti 41BB (CD137) APC309810 for receptor IgG1 400122 staining anti CD47 Alexa 647 MCA2514A647BioRad IgG2b MCA691A647 Antibodies used anti 41BBL PE 311504 Biolegendfor Binding IgG1, K 400112 assay

Methods—

For expression assays, cells (0.5 M cells/sample) were immuno-stainedwith the indicated antibodies, followed by Flow cytometry analysis. Forbinding assays, cells were pre-incubated with human Fc blocker prior toincubation with different concentrations (0.01-50 g/ml) of theHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) for 30 minutes on ice,followed by immuno-staining with antibodies against the “free” arm ofthe molecule (41BBL), fixation and analysis by flow cytometry.

Results—

As shown in FIGS. 4A-B, CHO-K1-WT cells do not express CD47 nor 41BB;while CHO-K1-CD47 cells express CD47 but do not express 41BB.

Binding assays showed that His-tagged SIRPα-41BBL (SEQ ID NO: 5) bindsto CHO-CD47 cells in as dose dependent manner, while it doesn't bind toCHO-WT cells (FIGS. 5A-B).

Taken together, the N terminal of His-tagged SIRPα-41BBL protein (SEQ IDNO: 5) can bind CD47 overexpressed on the surface of cells.

Experiment 2—Activation of the 41BB Receptor by the Chimera

The activation effect of the 41BB receptor by the His-tagged SIRPα-41BBLis tested by using HT1080 cells or another cell line that areoverexpressing the 41BB receptor. Specifically, the HT1080-41BB cellline is overexpressing 41BB and is known to secrete IL-8 upon binding of41BBL (Wyzgol, et al, 2009, The Journal of Immunology). Upon binding of41BBL to the 41BB receptor on the surface of these cells, a signalingpathway is activated resulting in secretion of IL8. The cells areincubated in the presence of the His-tagged SIRPα-41BBL in differentconcentrations and IL8 secretion to the culture media is determined byELISA. The oligomerization is tested by addition of anti-His-tag crosslinking antibody in different concentrations. With the addition of theanti-His-tag Ab, the chimera molecules will be cross linked and formoligomers, resulting in an increased IL8 secretion. Anti SIRPα antibodycan be used for the same purpose as well (cross linking the SIRPα moietyof the molecule).

The oligomerization is also tested by co-culturing the cellsoverexpressing the 41BB receptor with HT1080 cells that areoverexpressing human CD47 or with cancer cell line that are highlyexpressing CD47. The SIRPα-41BBL binds to the CD47 that is expressed onthe HT1080 or cancer cells and the 41BBL moiety is presented to theHT1080 that are overexpressing the 41BB receptor. Due to thispresentation of several molecules in close vicinity, the requirement foroligomerization is fulfilled.

The activation of the 41BBL receptor by His-tagged SIRPα-41BBL can becompared to that of its parts, namely, recombinant SIRPα or 41BBL aloneor in combination.

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove, HT1080-41BB cells (Lang et al 2015), IL-8ELISA kit (cat#D8000C, R&D), DMEM (cat#01-055-1A, Biologicalindustries), FBS (cat#10270106, Rhenium), AIM V (serum free medium)(ThermoScientific).

Methods—

HT1080-41BB cells (5000 per well) were incubated for 24 hours withdifferent concentrations of His-tagged SIRPα-41BBL protein (SEQ ID NO:5). IL-8 concentration in the supernatant was determined by IL-8 ELISAkit according to the manufacturer's protocol. Serum free medium was usedfor some of the experiments to eliminate relatively high background thatwas detected using medium with FBS.

Results—

Several independent experiments showed the functionality of SIRPα-41BBL:His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) was able to trigger TNFRsignaling as determined by IL8 secretion by HT1080-41BBL cells, in adose dependent manner both in medium containing FBS (FIG. 6) and inSerum free medium (FIG. 7).

Experiment 3—Activation of T-Cells by SIRPα-41BBL

The effect of SIRPα-41BBL on the activation of T-cells is tested usingeither T-cells in human healthy donor PBMCs or by using human TILs. TheT-cells are first co-cultured with human carcinoma cancer cells andtreated with anti CD3 and anti Epcam1 bispecific antibodies to induceT-cell activation and then with the SIRPα-41BBL. The anti CD3/Epcam1antibody is delivering the first signal for activation of T cellsagainst the Epcam1 expressing cancer cells. The SIRPα-41BBL molecule isinteracting with CD47 expressed on the surface of cancer cells, thisinteraction facilitates the presentation and oligomerization of themolecule and by that, enables the interaction of the 41BBL moiety with41BB receptor on The T cell and delivery of a second co-stimulatorysignal to the T cell. The activation level of the T cells is determinedby measuring several parameters; Firstly, by testing the expression ofactivation markers on the surface of the T cells, (for example: CD25,CD69, CD62L, CD137, CD107a, PD1 etc.). Expression of activation markersis tested by staining the cells with specific antibodies and flowcytometry analysis (FACS). A second way to determine T cell activationis by measuring inflammatory cytokine secretion (for example: IL2, IL6,IL8, INF gamma etc.). Secretion of inflammatory cytokine is tested byELISA. Proliferation of T cells is measured by pre-staining of T cellswith CFSE (carboxyfluorescein succinimidyl ester) and determiningdeviation of cells by CFSE dilution that is determined by FACS. Anadditional parameter that is tested is the killing of the cancer cellsthat is measured by pre-labeling the cancer cells using Calcine-AMreagent and measuring Calcine release into the culture medium usingluminescence plate reader.

The effect of SIRPα-41BBL on the activation of TILs is tested on TILsthat are extracted from tumors and then co-cultured with the tumorcancer cells and treated with SIRPα-41BBL. The first signal foractivation of T cells is delivered by the cancer cells via MHC class I:peptide—TCR (T cell receptor) pathway. The SIRPα-41BBL fusion protein isinteracting with CD47 expressed on the surface of the tumor cells, thisinteraction facilitates the presentation and oligomerization of themolecule and accordingly enables the interaction of the 41BBL moietywith 41BB receptor on the T cell and delivery of a second co-stimulatorysignal to the T cell. Activation level of the TILs and killing of tumorcells is determined in the same way as described (activation markers,cytokine secretion, proliferation and killing of tumor cells).

The activation of T-cells by His-SIRPα-41BBL can be compared to that ofits parts, namely, recombinant SIRPα or 41BBL alone or in combination.

Experiment 3A—SIRPα-41BBL Protein Demonstrates T Cell Co-StimulatoryActivity

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove; HT1080-41BB, CHO-WT, CHO-K1-CD47 and DLD1cell lines (Bommel et al 2017, Lang et al 2015, ATCC-CCL-221), freshlyisolated human T cells, IL8 Elisa kit (R&D systems, cat# DY208), CD47:FC(Sino Biological, cat #12283-HO2H), Anti-CD3/anti-CD28 activation beads(Life Technologies, cat#11131D), Anti CD25 antibody (Immuno Tools,cat#21270256), Lymphoprep (Stemcell, 07851) Name: CD14 MicroBeads, human(miltenyi Biotec, 130-050-201).

Methods and Results—

Upon treatment of single cultures of HT1080 cells transduced with 41BB(HT1080-41BB) with His-tagged SIRPα-41BBL (SEQ ID NO: 5), minimalproduction of IL-8 was detected following 24 hours of incubation (FIG.8A). Similarly, treatment with His-tagged SIRPα-41BBL (SEQ ID NO: 5)minimally induced IL-8 secretion when HT1080-41BB cells were mixed withwild-type CHO cells (FIG. 8A). However, treatment with His-taggedSIRPα-41BBL (SEQ ID NO: 5) of mixed cultures of HT1080-41BB withCHO-CD47 (enabling the SIRPα domain to bind to CHO-CD47 and presentcross-linked 41BBL to HT1080-41BB), triggered a strong increase in IL-8secretion that peaked at 2000 pg/mL (FIG. 8B). Thus, binding of theHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) to CD47 is beneficial inorder to stimulate IL-8 secretion upon 41BBL/41BB interaction.

Next, the potential induction of T cell activation by the 41BBL domainof His-tagged SIRPα-41BBL (SEQ ID NO: 5) was evaluated. PBMCs wereisolated from blood of healthy donors with Lymphoprep according tomanufacturer's instructions. Cells were then stained with CD14MicroBeads and T cells were isolated according to manufacturer'sinstructions with the MACS sorting system. To this end, freshly isolatedT cells were added to CD47-Fc coated plates and activated withsub-optimal concentrations of anti-CD3/anti-CD28 activation beads for 3days. Following treatment, a clear increase in the percentage ofactivated CD25+ T cells was detected in the His-tagged SIRPα-41BBLtreated cells (FIG. 8C), with an optimum induction at ˜2.5 μg/ml. Insubsequent mixed cultures of DLD-1 cells and T cells, the treatment withHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) increased the percentageof CD25+ T cells (FIGS. 8C-E), indicating that SIRPα-41BBL protein canactivate T cells. Thus, binding of His-tagged SIRPα-41BBL protein (SEQID NO: 5) to CD47 enables 41BBL/41BB-mediated co-stimulation andactivation of T cells.

Taken together, these data provide clear evidence that uponCD47-mediated binding, His-tagged SIRPα-41BBL protein (SEQ ID NO: 5)gains 41BBL-mediated co-stimulatory activity that can augment T cellactivation.

Experiment 3B—SIRPα-41BBL Protein Augments Human PBMCs Activation

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove; INF-γ ELISA Kit [cat#900-TM27,cat#900-T00—Elisa Buffer Kit (TMB)], RPMI (cat#01-100-1A, Biologicalindustries), FBS (cat#12657-029, Gibco), L-Glutamine (cat#25030-24,Gibco), Pen/Strep (cat#15140-122, Gibco), Leaf purified Anti-human CD3(cat# BLG-317315, BioLegend), Recombinant human IL2 (cat#202-IL-500, R&DSystems), Human Peripheral Blood Mononuclear Cells (PBMCs) isolated fromhealthy donor peripheral blood by Ficoll-Paque (cat#17-1440-03, GEHealthcare), anti-CD47 antibody (cat#MCA2514A647, Biorad), anti-41BBLantibody (cat#311504, Biolegend), MV4-11 human leukemia cells (ATCC,Abraham et al 2017).

Methods—

MV4-11 cells were tested for CD47 expression and binding of His-taggedSIRPα-41BBL by flow cytometry. Human PBMCs were isolated from healthydonor peripheral blood using Ficoll-Paque method (Grienvic et al. 2016).Following, PBMCs were cultured for 40 hours with addition of differentconcentrations of His-tagged SIRPα-41BBL protein (SEQ ID NO: 5), in thepresence of anti-CD3 (30 ng/ml) or anti-CD3 plus IL2 (1000 U/ml). Theexperiment was effected with or without co-culture with CD47 expressinghuman cancer cell line MV4-11 (ratio 1:1). INF-γ concentration in thecells supernatant was determined by INF-γ ELISA kit according to themanufacturer's protocol.

Results—

Human PBMCs, including NK cells, NKT cells, CD4+ and CD8+ effectorcells, are known to secrete pro-inflammatory Interferon-γ (INF-γ) inresponse to activation. The activation of a T cell requires two signals:ligation of the T-Cell Receptor (TCR) with the Major HistocompatibilityComplex (MHC)/peptide complex on the Antigen Presenting Cell (APC) andcross-linking of co-stimulatory receptors on the T cell with thecorresponding ligands on the APC. 41BB, is a T cell co-stimulatoryreceptor induced by ligation of 41BBL. 41BB transmits a potentcostimulatory signal to both CD8+ and CD4+ T cells, promoting theirexpansion, survival, differentiation, and cytokine expression. Itsligand, 41BBL, is a membrane protein, which provides a co-stimulatorysignal to T cells.

In this experiment the functionality of SIRPα-41BBL molecule inenhancing human PBMCs activation was evaluated.

CD47 is present on the surface of MV4-11 cells and His-taggedSIRPα-41BBL protein (SEQ ID NO: 5) bound these cells in a dose dependentmanner (FIGS. 9A-B). Incubation of MV4-11 cells with differentconcentrations of His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) up to 72hours did not show any direct killing effect (FIG. 9C).

Addition of His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) enhanced theactivation of PBMCs in a dose depended manner, as can be seen by anincrease in INF-γ secretion by PBMCs that were stimulated with anti-CD3antibody, with or without the addition of IL2 (FIG. 10A).

Co-culturing PBMCs with human cell line MV4-11 and stimulating the cellswith anti CD3 antibody resulted in INF-γ secretion, probably due todirect stimulation of the PBMCs cells by the MV4-11 cells. Treatmentwith His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) had a moderate effectthat was more pronounced when added together with IL2 (FIG. 10B).

Taken together, His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) augmentsactivation of human PBMCs, as can be seen by increase in IFN-γ secretion

Experiment 4—Effect of SIRPα-41BBL on Granulocytes and Macrophages

SIRPα is an inhibitory receptor expressed on the cell surface of e.g.phagocytic cells. CD47, the ligand for SIRPα, is extensively expressedon tumor cells. Upon engaging of SIRPα by CD47, SIRPα delivers a “don'teat me” signal to phagocytic cells. By interaction of SIRPα-41BBL withCD47, it should block the endogenous interaction of CD47 with SIRPα andby that block the “don't eat me” signal, allowing the engulfment of thetumor cells by phagocytic cells.

The effect of SIRPα-41BBL on granulocytes, macrophages and otherphagocytic cells, is tested in-vitro in a co-culture assay usinggranulocytes or M1 macrophages from healthy donors co-cultured withfluorescently labeled CD47 expressing cancer cells. SIRPα-41BBL is addedto the co-culture in different concentrations and phagocytosis isdetermined by measuring the fluorescent uptake by the granulocytes or M1macrophages, using flow cytometry. The phagocytic cells are identifiedand distinguished from the cancer cells by staining for specific surfacemarkers (like CD11b).

The phagocytic effect in this experiment can be enhanced usingTherapeutic Anti-Tumor antibodies (such as Rituximab, Cetuximab,Trastuzumab, Alemtuzumab, etc.) Similar experiment is done usingautologous granulocytes from cancer patient co-cultured with primaryautologous malignant cells as well.

Materials—

His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) produced as described inExperiment 1A hereinabove, Human leukocytes isolated from peripheralblood, tumor cell lines: BJAB, U2932, Raji, Sudhl6, DLD1, H292, FADU,OVCR, MOLM13, K562, OCIAML3, HL60 (ATCC), vybrant DiD (Invitrogen, cat#V22887), cell proliferation dye V450 (Thermofishr, 65-0842-85),Lymphoprep (Stemcell technology, 07851) Recombinant human SIRPα (sinobiological, 11612-H08H) Recombinant human 41BBL (R&D systems,2295-4L/CF).

Methods and Results—

Leukocytes were isolated from peripheral blood of healthy donors. Inshort, whole blood was mixed with PBS containing EDTA at ratio of 1:1and mixed with lymphoprep. To obtain neutrophils, PBMCs were removedfollowing lymphoprep and cell pellet was harvested. Cell pellet wasmixed with erythrocytes lysis buffer at a ratio of 1:10 and incubated at4° C. for 30-45 minutes. Following, neutrophils were harvested bycentrifugation at 450 g/5 minutes and washed twice with PBS containingEDTA Isolated leukocytes were mixed with tumor cells, that had beenpre-stained with vybrant DiD, at a 1:1 ratio. The uptake of tumor cellsby granulocytes was subsequently evaluated using flow cytometry (forgating strategy see FIG. 11A). In such mixed cultures, granulocytesminimally phagocytose tumor cells in the absence of any stimulus (FIG.11A). However, when mixed cultures of leukocytes and Ramos B-NHL cellswere treated with increasing concentration of His-tagged SIRPα-41BBL(SEQ ID NO: 5), a clear dose-dependent induction of phagocytosis bygranulocytes was detected, with an optimum effect achieved at 2.5 μg/ml(FIGS. 11A-B). Similar pro-phagocytic activity of single agent treatmentwith His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) was detected toward apanel of B-cell lymphoma cell lines, yielding an increase inphagocytosis of 15% to 20% compared to untreated cultures (FIG. 11C).

Following, the mixed cultures were treated with a combination ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) and the anti-CD20 antibodyrituximab. As shown in FIG. 11D, treatment with low sub-optimalconcentration of rituximab already triggered phagocytosis bygranulocytes, the extent of which depended on the leukocytes donor andcell line (FIG. 11D, white diamond squares). However, combinationtreatment with rituximab and His-tagged SIRPα-41BBL protein (SEQ ID NO:5) further increased phagocytic uptake of all B-NHL cell lines tested(FIG. 11D, black diamond squares).

To determine whether combination of SIRPα-41BBL with other therapeuticantibodies would similarly augment phagocytosis, a panel of carcinomacell lines was mixed with leukocytes. Phagocytosis by granulocytes wasevaluated upon treatment with His-tagged SIRPα-41BBL protein (SEQ ID NO:5) with or without the anti-EGFR antibody cetuximab. In all carcinomacell lines tested, single agent treatment with the His-taggedSIRPα-41BBL already strongly triggered phagocytic uptake of cancer cells(FIG. 11E), with up to 80% of granulocytes phagocytosing DLD-1 cells.Upon combination treatment with His-tagged SIRPα-41BBL and cetuximab,the phagocytic uptake of cancer cells was increased even further (FIG.11F): for example, in the squamous cell carcinoma line FaDu, over 90%were phagocytosed, suggesting an additive or even synergistic effect ofcetuximab with SIRPα-41BBL for this but also other cell lines.

Subsequent analysis of phagocytosis of several acute myeloid leukemiacell lines revealed that most of these cell lines did not respond totreatment with His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) alone (FIG.11G). A notable exception here was HL60, an acute promyelocytic leukemiacell line, with an increase in HL60 phagocytosis of ˜10-20% (dependingon the donor) compared to untreated cultures, following 2 hours ofincubation. Upon prolonged treatment of up to 24 hours,granulocyte-mediated phagocytosis of K562 (chronic myelogenous leukemia)and Oci-AML3 (acute myeloid leukemia) was also strongly increasedcompared to untreated cultures, whereas HL60 uptake at this extendedtime-point was already very high in the untreated control cultures (FIG.11H). Increasing the His-tagged SIRPα-41BBL (SEQ ID NO: 5) dose at the 2hours' time-point further increased phagocytosis of HL60, but not ofMOLM13 (FIG. 11I).

Importantly, also primary AML blasts, obtained from an AML patient withcomplex karyotype, were phagocytosed upon treatment with His-taggedSIRPα-41BBL (SEQ ID NO: 5) in a dose-dependent manner (FIG. 11J). In anextended panel of allogeneic granulocytes from five healthy donors,these primary AML blasts were strongly phagocytosed within 2 hours witha median increase in phagocytosis of ˜35% compared to untreated cultures(FIG. 11K). Moreover, following 24 hours the phagocytosis in untreatedcultures was increased, but was still enhanced upon His-taggedSIRPα-41BBL (SEQ ID NO: 5) treatment (FIG. 11K).

To further establish the potential of SIRPα-41BBL to stimulatephagocytosis, similar experiments were performed using monocyte-derivedmacrophages. Shortly, monocytes were enriched from isolated PBMCs fromhealthy donors by MACS sorting using CD14 magnetic MicroBeads (MiltenyiBiotec). Monocytes were differentiated into macrophages (M0) in RPMI1640 culture medium+10% FCS supplemented with GM-CSF (50 ng/ml) andM-CSF (50 ng/ml) for 7 days. To generate type 1 macrophages, M0 cellswere primed by LPS and IFN-γ for additional 24 hours. Following, the Invitro differentiated macrophages were mixed with B-cell lymphoma cellline U2932 that was pre-stained with the cell proliferation dye V450.Macrophages were mixed with U2932 for 2 hours; and phagocytic uptake ofcancer cells was determined by fluorescent microscopy (representativemicroscopy pictures are shown in FIG. 12A, with dark arrows indicatingviable cancer cells and white arrows indicating phagocytosed cancercells in macrophages). Treatment with His-tagged SIRPα-41BBL (SEQ ID NO:5) had a limited activity in this setting (FIG. 12B). Treatment withrituximab induced a strong increase in of phagocytosis (FIG. 12C, whitediamond squares), which was further augmented by co-treatment withHis-tagged SIRPα-41BBL (SEQ ID NO: 5) in the majority of donors atvarying rituximab concentrations (FIG. 12C, black diamond squares).

Further, in a mixed culture experiment with primary B-cell chroniclymphocytic leukaemia (B-CLL) blasts and autologous patient-derivedmacrophages, treatment with the anti-CD52 antibody alemtuzumab (as theseblasts were highly positive for CD52) alone induced macrophage-inducedphagocytosis of nearly 40% (FIG. 11D); treatment with His-taggedSIRPα-41BBL (SEQ ID NO: 5) alone had a minimal pro-phagocytic activity;but in this primary sample the combination of His-tagged SIRPα-41BBL(SEQ ID NO: 5) and alemtuzumab augmented phagocytosis (by ˜20% ascompared to treatment with alemtuzumab) (FIG. 12D).

Moreover, the effect of the his-tagged SIRPα-41BBL fusion protein (SEQID NO: 5) on phagocytes was superior in comparison to the effects ofsoluble SIRPα alone, soluble 41BBL alone, or their combination (FIG.11L).

Taken together, His-tagged SIRPα-41BBL (SEQ ID NO: 5) through its SIRPαdomain augments both granulocyte and macrophage-mediated phagocyticuptake of various malignant cell types, including primary malignantcells, particularly in combination treatment with various therapeuticmonoclonal antibodies currently in clinical use.

Experiment 5—In-Vivo Proof of Concept

The effects of SIRPα-41BBL, both on the targeting and activation of T,NK and B cells and on the activation of phagocytic and dendritic cells,is tested in-vivo in mouse models. The mouse His-tagged SIRPα-41BBLfusion protein is produced and purified as tagged protein in the sameway as the human molecule. Mouse tumor models are generated by injectingmice with mouse cancer cells that are known to form tumors that expressmouse CD47. Mice are treated with the mouse or humanHis-tagged-SIRPα-41BBL fusion protein molecule. Tumor size, micesurvival and inflammatory reaction in the tumor site are monitored.

Similar experiments can be performed in a humanized mouse model usinghuman tumors. This model is constructed using mice that are lacking anymouse immune system (Nude/SCID/NSG mice). A human-like immune system isestablished in these mice by injection of only human T cells or PBMCs orby using genetically engineered mice that possess a fully humanizedimmune system. The mice are inoculated with human cancer cells andtreated with the human His-tagged SIRPα-41BBL molecule. Tumor size, micesurvival and inflammatory reaction in the tumor site are monitored inthis model as well.

The in-vivo efficacy of His-SIRPα-41BBL can be compared to that of itsparts, namely, recombinant SIRPα or 41BBL alone or in combination.

Experiment 5A—SIRPα-41BBL protein inhibits tumor growth in miceinoculated with syngeneic colon carcinoma

Materials—

Mice autoclaved food and bedding (Ssniff, Soest, Germany), Female Balb/Cmice (Janvier, Saint Berthevin Cedex, France), CT-26 mouse coloncarcinoma cell line (ATCC-CRL-2638), anti-mouse PD-1 antibody (BioXcell,West Lebanon, USA), His-tagged SIRPα-41BBL protein (SEQ ID NO: 5)produced as described in Experiment 1A hereinabove, PBS

Methods—

Mice were maintained in individually ventilated cages in groups of fourmice per cage. The mice received autoclaved food and bedding andacidified (pH 4.0) tap water ad libitum. The animal facility wasequipped with an automatic 12 hours light/dark regulation, temperatureregulation at 22±2° C., and relative humidity of 50±10%. Female Balb/Cmice were inoculated subcutaneously with 1×10⁶ CT-26 cells and treatmentstarted three days later. Following random assignment, 10 animals pergroup were administered twice weekly with three intravenous injectionsof His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) (100 μg/injection) orits soluble buffer (PBS). 5 mg/kg anti-mouse PD1 at the same schedulewere included as a therapeutic reference (FIG. 13A). All administrationswere performed in the morning, without anesthesia. Tumor volume wasdetermined three times per week using caliper measurements, and theindividual volumes were calculated by the formula:V=([width]2×length)/2. All animal experiments were done in accordancewith the United Kingdom Coordinating Committee on Cancer Researchregulations for the Welfare of Animals (Workman et al., Committee of theNational Cancer Research Institute. Guidelines for the welfare ofanimals in cancer research. Br J Cancer 2010; 102:1555-77) and of theGerman Animal Protection Law and approved by the local responsibleauthorities (Gen0030/15).

Results—

In this Experiment, the in-vivo effect of SIRPα-41BBL was evaluatedusing the CT-26 mouse colon cancer model. Treatment of CT-26 inoculatedmice with His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) reduced thetumor volume (by about 36% at max) (FIG. 13B).

Experiment 5B—SIRPα-41BBL Protein is Effective for the Treatment of MiceInoculated with a Syngeneic Leukemic Tumor

Materials—

Mice autoclaved food and bedding (Ssniff, Soest, Germany), Female DBA/2mice (Janvier, Saint Berthevin Cedex, France), P388 Leukaemia cell line(Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany),anti-mouse PD-1 antibody (BioXcell, West Lebanon, USA), His-taggedSIRPα-41BBL protein (SEQ ID NO: 5), PBS

Methods—

Mice were maintained in individually ventilated cages in groups of fourmice per cage. They received autoclaved food and bedding and acidified(pH 4.0) tap water ad libitum. The animal facility was equipped with anautomatic 12 hours light/dark regulation, temperature regulation at22±2° C., and relative humidity of 50±10%. Female DBA/2 mice wereinoculated intraperitoneally with 1×10⁶ P388 cells and treatment startedthe day after. Following random assignment, ten animals per group wereadministered every second day with four intravenous injections ofHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) (100 μg/injection) or itssoluble buffer (PBS). 5 mg/kg anti-mouse PD1 at the same schedule wereincluded as a therapeutic reference. All administrations were performedin the morning, without anesthesia. Mice bearing P388 were weighed dailyand once the mice became moribund, they were sacrificed, and the ascitesvolume was determined. Furthermore, spleen and liver from each mouse wastaken and weighed.

All animal experiments were done in accordance with the United KingdomCoordinating committee on Cancer Research regulations for the Welfare ofAnimals (Workman et al., committee of the National Cancer ResearchInstitute. Guidelines for the welfare of animals in cancer research. BrJ Cancer 2010; 102:1555-77) and of the German Animal Protection Law andapproved by the local responsible authorities (Gen0030/1

Results—

In this experiment, the in-vivo effect of SIRPα-41BBL was evaluatedusing the P388 mouse ascites leukemia model. In this model, spleenweight is a marker for disease severity, due to the fact that the spleenserves as draining lymph node for the ascites. Treatment of P388 mouseleukemia inoculated mice with His-tagged SIRPα-41BBL protein (SEQ ID NO:5) was effective, as can be seen by the significant reduction in spleenweight (19%, P-value=0.03) (FIG. 14B) pointing of a better diseaseprognosis.

Experiment 5C—SIRPα-41BBL Protein Decreases Tumor Burden in the BM ofNSG Mice Inoculated with Human Leukemia Tumor

Materials—

Female NSG (NOD-scid gamma mice) mice at the age of 12-16 weeks (JacksonLaboratory), MV4.11 Acute Myeloid Leukemia cell-line (ATCC-CRL-9591),anti-mouse CD45 antibody (eBioscience, San Diego, Calif., USA),anti-human CD45 antibody (eBioscience, San Diego, Calif., USA),anti-human CD123 antibody (BD Pharmingen, USA), His-tagged SIRPα-41BBLprotein (SEQ ID NO: 5) produced as described in Experiment 1Ahereinabove, PBS. Human PBMCs were isolated from healthy donorperipheral blood using Ficoll-Paque method (Sigma-Aldrich Israel Ltd.).

Methods—

Mice were maintained in individually ventilated cages in groups of 5mice per cage. The mice received autoclaved food and bedding andacidified (pH 4.0) tap water ad libitum. The animal facility wasequipped with an automatic 12 hours light/dark regulation, temperatureregulation at 22±2° C., and relative humidity of 50±10%. Female NSG micewere irradiated with 200 rad 24 hour prior to intravenous inoculationthrough the tail vein with 7.8×10⁶ MV4.11 cells in a total volume of 200al PBS. Thirteen (13) days later mice were inoculated through the tailvein with 1.5×10⁶ human PBMCs and treatment started 4 hours later.Following random assignment, 5 animals per group were administeredevery-other-day (EOD) with four intraperitoneal injections of His-taggedSIRPα-41BBL protein (SEQ ID NO: 5, 100 μg/injection) or its solublebuffer (PBS) (on days 13, 15, 17 and 19) (FIG. 15A). All administrationswere performed in the morning, without anesthesia. Twenty-four (24)hours following last injection mice were sacrificed and blood,bone-marrow (BM) and spleen were collected. Spleen was weighted, and BMextracted cells were analyzed by FACS following staining with hCD45,mCD45 and CD123.

All animal experiments were done in accordance with the Hadassa MedicalCenter Committee regulations for the Welfare of Animals and of theIsraeli Animal Protection Law and approved by the local responsibleauthorities.

Results—

Treatment of MV4.11 inoculated irradiated NSG mice not reconstitutedwith human PBMCs with His-tagged SIRPα-41BBL protein (SEQ ID NO: 5) didnot affect the number of leukemic cells in the BM (FIG. 15B). Treatmentof MV4.11 inoculated NSG mice with human PBMCs reduced tumor burden inthe bone marrow (by 10%) (FIG. 15B) and did not increase spleen weight(FIG. 15C). However, Treatment of MV4.11 inoculated NSG mice withHis-tagged SIRPα-41BBL protein (SEQ ID NO: 5) together with human PBMCsreduced tumor burden in the bone marrow (by 38%) (FIG. 15B) and alsoincreased spleen weight (FIG. 15C). The Increase in spleen weight thatwas seen only with the combination of His-tagged SIRPα-41BBL protein andhuman PBMCs is indicative of increased graft versus leukemia (GVL)effect in the BM that was also associated with reduction of leukemiacell number in the BM (FIGS. 15B-C, p value=0.01).

1. A SIRPα-41BBL fusion protein being characterized by a single aminoacid linker between said SIRPα and said 41BBL; and/or being in a form ofat least a homo-trimer.
 2. (canceled)
 3. The SIRPα-41BBL fusion proteinof claim 1, wherein said at least homo-trimer is at least 140 kD inmolecular weight as determined by SDS-PAGE.
 4. The SIRPα-41BBL fusionprotein of any one of claim 1, wherein the SIRPα-41BBL fusion proteincomprises a linker between said SIRPα and said 41BBL. 5-6. (canceled) 7.The SIRPα-41BBL fusion protein of claim 4, wherein the linker is not anFc domain of an antibody or a fragment thereof.
 8. The SIRPα-41BBLfusion protein of any one of claim 1, wherein the linker is glycine. 9.The SIRPα-41BBL fusion protein of claim 1, being soluble. 10-11.(canceled)
 12. The SIRPα-41BBL fusion protein of claim 1, wherein saidfusion protein is capable of at least one of: (i) binding CD47 and 41BB;(ii) activating said 41BB signaling pathway in a cell expressing said41BB; (iii) co-stimulating immune cells expressing said 41BB; and/or(iv) enhancing phagocytosis of pathologic cells expressing said CD47 byphagocytes compared to same in the absence of said SIRPα-41BBL fusionprotein.
 13. The SIRPα-41BBL fusion protein of claim 1, wherein saidSIRPα-41BBL fusion protein amino acid sequence comprises SEQ ID NO: 1.14. The SIRPα-41BBL fusion protein of claim 1, wherein said SIRPα-41BBLfusion protein amino acid sequence consists of SEQ ID NO:
 1. 15. Apolynucleotide encoding the SIRPα-41BBL fusion protein of claim
 1. 16. Anucleic acid construct comprising the polynucleotide of claim 15, and aregulatory element for directing expression of said polynucleotide in ahost cell.
 17. (canceled)
 18. A host cell comprising the SIRPα-41BBLfusion protein of claim
 1. 19. A method of producing a SIRPα-41BBLfusion protein, the method comprising expressing in a host cell thepolynucleotide of claim
 15. 20. The method of claim 19, comprisingisolating the fusion protein. 21-22. (canceled)
 23. A method of treatinga disease that can benefit from activating immune cells comprisingadministering to a subject in need thereof the SIRPα-41BBL fusionprotein of claim
 1. 24. An article of manufacture identified for thetreatment of a disease that can benefit from activating immune cellscomprising a packaging material packaging a therapeutic agent fortreating said disease; and a SIRPα-41BBL fusion protein, apolynucleotide encoding same, a nucleic acid construct encoding same ora host cell expressing same.
 25. The method of claim 23, wherein saiddisease comprises a hyper-proliferative disease, a disease associatedwith immune suppression or medication induced immunosuppression, or aninfection.
 26. (canceled)
 27. The method of claim 25, wherein saidhyper-proliferative disease comprises cancer.
 28. A method of treatingcancer comprising administering to a subject in need thereof ananti-cancer agent; and a SIRPα-41BBL fusion protein, a polynucleotideencoding same, a nucleic acid construct encoding same or a host cellexpressing same.
 29. The method of claim 27, wherein said cancer isselected from the group consisting of lymphoma, leukemia, colon cancer,pancreatic cancer, ovarian cancer, lung cancer and squamous cellcarcinoma.
 30. The method of claim 27, wherein cells of said cancerexpress CD47. 31-33. (canceled)
 34. The method of claim 23, whereindiseased cells of said subject express CD47. 35-36. (canceled)
 37. Amethod of activating immune cells, the method comprising in-vitroactivating immune cells in the presence of a SIRPα-41BBL fusion protein,a polynucleotide encoding same, a nucleic acid construct encoding sameor a host cell expressing same.
 38. The method of claim 37, wherein saidactivating is in the presence of cells expressing CD47 or exogenousCD47.
 39. The method of claim 38, wherein said cells expressing saidCD47 comprise pathologic cells. 40-41. (canceled)
 42. The method ofclaim 37, wherein said activating is in the presence of an anti-canceragent.
 43. The method of claim 28, wherein said anti-cancer agentcomprises an antibody.
 44. (canceled)
 45. The method of claim 43,wherein said antibody is selected from the group consisting ofrituximab, cetuximab and almetuzumab.
 46. The method of claim 27,comprising adoptively transferring said immune cells following saidactivating to a subject in need thereof.
 47. (canceled)
 48. The methodof claim 28, wherein said SIRPα-41BBL fusion protein is characterized bya single amino acid linker between said SIRPα and said 41BBL; and/orbeing in a form of at least a homo-trimer. 49-50. (canceled)